Egfr assay

ABSTRACT

Provided herein is technology relating to detecting molecular markers relevant to cancer and particularly, but not exclusively, to methods and compositions for quantifying and/or detecting EGFR mRNA and/or EGFRvIII mRNA in biological samples.

This application is a continuation of U.S. patent application Ser. No.15/191,122, filed Jun. 23, 2016, which claims priority to U.S.provisional patent application Ser. No. 62/183,566, filed Jun. 23, 2015,which is incorporated herein by reference in its entirety.

FIELD

Provided herein is technology relating to treating cancer andparticularly, but not exclusively, to methods and compositions forquantifying and/or detecting EGFR and/or

EGFRvIII in biological samples.

BACKGROUND

The Epidermal Growth Factor Receptor (EGFR) gene is frequently amplifiedin a variety of cancer tumors. In some cancers, the resultingover-expression of EGFR mRNA and/or protein activates downstream signaltransduction pathways, which contributes to cell cycle dysregulation andtumorigenesis. In addition to over-expressing wild-type EGFR (wtEGFR),some tumor cells also express a rearranged form of EGFR in which exons 2through 7 are deleted, thereby juxtaposing exons 1 and 8. This mutantEGFR is known as EGFRde2-7 or EGFR mutant variant III (EGFRvIII). TheEGFRvIII protein product induces ligand-independent cell signaling,which further increases the tumorigenic potential of cells expressingthe mutation.

Anti-EGFR therapies are designed to target and destroy cancer cells thatoverexpress EGFR and/or express EGFRvIII. Thus, assessment of EGFRand/or EGFRvIII RNA expression profiles may serve as importantdiagnostic tools in the field of oncology.

SUMMARY

Provided herein is a technology for single-well detection of RNA (e.g.,EGFRvIII RNA, EGFR RNA, and/or an endogenous control RNA such asbeta-actin (ACTB) RNA, abelson tyrosine kinase (ABL) RNA, and/orglucose-6-phosphate (G6PD) RNA) and/or for single-well detection of acDNA (e.g., a cDNA produced from an EGFRvIII RNA, an EGFR RNA, and/orfrom an endogenous control RNA such as ACTB RNA, ABL RNA, and/or G6PDRNA). For example, some embodiments of the technology are related to adiagnostic method and assay format that provide specific and sensitivedetection of EGFRvIII and quantification of total EGFR mRNA expressionusing real-time RT-PCR technology (e.g., producing an EGFRvIII cDNAand/or an EGFR cDNA by reverse transcription, then amplifying the cDNA).In some embodiments, the technology provides a single-well multiplexRT-PCR for the detection of EGFRvIII expression (e.g., detection ofEGFRvIII mRNA and/or cDNA), quantification of EGFR expression (e.g.,quantification of total EGFR mRNA and/or cDNA, e.g., quantification ofwild-type EGFR mRNA and/or cDNA and EGFRvIII mRNA and/or cDNA), and/ordetection and/or quantification of the expression of an endogenouscontrol such as ACTB, ABL, and/or G6PD (e.g., detection and/orquantification of ACTB, ABL, and/or G6PD mRNA and/or cDNA). Accordingly,the technology provides in some embodiments a diagnostic and/orprognostic tool for oncology and related fields.

Embodiments of the technology include an assay format that detects mRNAfrom an endogenous control (e.g., a housekeeping gene such asbeta-actin), which is used to facilitate the quantification of EGFRvIIIexpression and total EGFR expression; and/or to provide a samplevalidity control for cell adequacy, sample extraction, and/oramplification efficiency. In some embodiments, the technology providesnovel primers and probes specific for EGFRvIII and total EGFR. Forexample, in some embodiments, the technology described hereinprovides: 1) a novel design for an oligonucleotide primer set thatdirects reverse transcription and amplification of EGFRvIII RNA; 2) anovel design for an oligonucleotide primer set that directs reversetranscription and amplification of total EGFR RNA (e.g., bothnon-rearranged wild type EGFR and EGFRvIII mutant); 3) a novel designfor a labeled oligonucleotide probe to detect EGFRvIII RNA; 4) a noveldesign for a labeled oligonucleotide probe to detect total EGFR RNA(both non-rearranged wild type EGFR and EGFRvIII mutant); and 5) a novelprimer/probe set for amplifying and detecting RNA sequences from anendogenous control (e.g., a house-keeping gene such as ACTB, ABL, and/orG6PD).

The EGFR primers and probe target the exon 29/30 junction, which ispresent in both wild-type EGFR mRNA and in the EGFRvIII variant mRNA(e.g., to target total EGFR mRNA, e.g., to quantify and/or to detecttotal EGFR mRNA). The EGFRvIII primers and probe target the exon 1/8junction (e.g., a target that is specific for EGFRvIII mRNA). Bothprimer/probe sets are designed to target exon junctions to detect RNAand/or cDNA, without detecting genomic DNA. Each amplicon is less than90 nt (e.g., to provide a technology that is appropriate for applicationto formalin-fixed paraffin-embedded (FFPE) samples).

Accordingly, provided herein are embodiments of a technology related toa composition for detecting EGFRvIII mRNA. In particular embodiments,the composition comprises a primer comprising a sequence according toSEQ ID NO: 1 or SEQ ID NO: 31 (e.g., a primer comprising a sequence or areverse complement of a sequence that is at least 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to SEQ ID NO: 1 or SEQ ID NO: 31 or that is 100%identical to SEQ ID NO: 1 or SEQ ID NO: 31) and a primer comprising asequence according to SEQ ID NO: 2 (e.g., a primer comprising a sequenceor a reverse complement of a sequence that is at least 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% identical to SEQ ID NO: 2 or that is 100% identical toSEQ ID NO: 2). In some embodiments, the composition for detectingEGFRvIII mRNA further comprises a detectably labeled probe comprising asequence according to SEQ ID NO: 3 (e.g., a probe comprising a sequenceor a reverse complement of a sequence that is at least 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% identical to SEQ ID NO: 3 or that is 100% identical toSEQ ID NO: 3).

Additional embodiments provide a composition for detecting and/orquantifying total EGFR mRNA. In particular embodiments, the compositioncomprises a primer comprising a sequence according to SEQ ID NO: 7(e.g., a primer comprising a sequence or a reverse complement of asequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical toSEQ ID NO: 7 or that is 100% identical to SEQ ID NO: 7) and a primercomprising a sequence according to SEQ ID NO: 8 (e.g., a primercomprising a sequence or a reverse complement of a sequence that is atleast 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 8 or thatis 100% identical to SEQ ID NO: 8). In some embodiments, the compositionfor detecting and/or quantifying total EGFR mRNA further comprises adetectably labeled probe comprising a sequence according to SEQ ID NO: 9or SEQ ID NO: 32 (e.g., a probe comprising a sequence or a reversecomplement of a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 9 or SEQ ID NO: 32 or that is 100% identical toSEQ ID NO: 9 or SEQ ID NO: 32).

Additional embodiments provide a composition for detecting and/orquantifying ACTB mRNA. In particular embodiments, the compositioncomprises a primer comprising a sequence according to SEQ ID NO: 13(e.g., a primer comprising a sequence or a reverse complement of asequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical toSEQ ID NO: 13 or that is 100% identical to SEQ ID NO: 13) and a primercomprising a sequence according to SEQ ID NO: 14 (e.g., a primercomprising a sequence or a reverse complement of a sequence that is atleast 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 14 or thatis 100% identical to SEQ ID NO: 14). In some embodiments, thecomposition for detecting and/or quantifying ACTB mRNA further comprisesa detectably labeled probe comprising a sequence according to SEQ ID NO:15 (e.g., a probe comprising a sequence or a reverse complement of asequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical toSEQ ID NO: 15 or that is 100% identical to SEQ ID NO: 15).

Some embodiments provide a composition for detecting EGFRvIII mRNA andfor quantifying total EGFR mRNA (e.g., in a multiplex, single-tubereaction). In particular embodiments, the composition comprises a primercomprising a sequence according to SEQ ID NO: 1 or SEQ ID NO: 31 (e.g.,a primer comprising a sequence or a reverse complement of a sequencethat is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1or SEQ ID NO: 31 or that is 100% identical to SEQ ID NO: 1 or SEQ ID NO:31), a primer comprising a sequence according to SEQ ID NO: 2 (e.g., aprimer comprising a sequence or a reverse complement of a sequence thatis at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 2 orthat is 100% identical to SEQ ID NO: 2), a primer comprising a sequenceaccording to SEQ ID NO: 7 (e.g., a primer comprising a sequence or areverse complement of a sequence that is at least 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to SEQ ID NO: 7 or that is 100% identical to SEQID NO: 7), and a primer comprising a sequence according to SEQ ID NO: 8(e.g., a primer comprising a sequence or a reverse complement of asequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical toSEQ ID NO: 8 or that is 100% identical to SEQ ID NO: 8). In someembodiments of the composition for detecting EGFRvIII mRNA and forquantifying total EGFR mRNA, the composition further comprises adetectably labeled probe comprising a sequence according to SEQ ID NO: 3(e.g., a probe comprising a sequence or a reverse complement of asequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical toSEQ ID NO: 3 or that is 100% identical to SEQ ID NO: 3) and a detectablylabeled probe comprising a sequence according to SEQ ID NO: 9 or SEQ IDNO: 32 (e.g., a probe comprising a sequence or a reverse complement of asequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical toSEQ ID NO: 9 or SEQ ID NO: 32 or that is 100% identical to SEQ ID NO: 9or SEQ ID NO: 32). And, in some embodiments of the composition fordetecting EGFRvIII mRNA and for quantifying total EGFR mRNA, thecomposition further comprises primers and probes for quantifying and/ordetecting a control mRNA such as ACTB. For example, some embodimentsprovide a composition for detecting EGFRvIII mRNA and for quantifyingtotal EGFR mRNA comprising a primer comprising a sequence according toSEQ ID NO: 13 (e.g., a primer comprising a sequence or a reversecomplement of a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 13 or that is 100% identical to SEQ ID NO: 13),a primer comprising a sequence according to SEQ ID NO: 14 (e.g., aprimer comprising a sequence or a reverse complement of a sequence thatis at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 14 orthat is 100% identical to SEQ ID NO: 14), and a detectably labeled probecomprising a sequence according to SEQ ID NO: 15 (e.g., a probecomprising a sequence or a reverse complement of a sequence that is atleast 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 15 or thatis 100% identical to SEQ ID NO: 15).

Particular embodiments related to multiplex detection provide acomposition wherein the detectably labeled probe comprising a sequenceaccording to SEQ ID NO: 3 (e.g., a probe comprising a sequence or areverse complement of a sequence that is at least 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to SEQ ID NO: 3 or that is 100% identical to SEQID NO: 3) comprises a first distinguishable fluorescent moiety, thedetectably labeled probe comprising a sequence according to SEQ ID NO: 9or SEQ ID NO: 32 (e.g., a probe comprising a sequence or a reversecomplement of a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 9 or SEQ ID NO: 32 or that is 100% identical toSEQ ID NO: 9 or SEQ ID NO: 32) comprises a second distinguishablefluorescent moiety, and the detectably labeled probe comprising asequence according to SEQ ID NO: 15 (e.g., a probe comprising a sequenceor a reverse complement of a sequence that is at least 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% identical to SEQ ID NO: 15 or that is 100% identical toSEQ ID NO: 15) comprises a third distinguishable fluorescent moiety,e.g., to detect each probe separately by monitoring a separate detectionchannel in a real-time PCR.

Some embodiments relate to detecting EGFRvIII mRNA and quantifying totalEGFR mRNA in a sample obtained from a subject, e.g., as a cancerdiagnostic. Thus, in some embodiments, compositions further comprise anucleic acid (e.g., RNA or DNA (e.g., cDNA)) prepared from a sampleobtained from a subject.

Some embodiments provide a composition comprising a primer consisting ofa sequence or a reverse complement of a sequence according to SEQ ID NO:1 or SEQ ID NO: 31 and a primer consisting of a sequence or a reversecomplement of a sequence according to SEQ ID NO: 2. In some embodiments,the composition for detecting EGFRvIII mRNA further comprises adetectably labeled probe consisting of a sequence or a reversecomplement of a sequence according to SEQ ID NO: 3.

Additional embodiments provide a composition for detecting and/orquantifying total EGFR mRNA. In particular embodiments, the compositioncomprises a primer consisting of a sequence or a reverse complement of asequence according to SEQ ID NO: 7 and a primer consisting of a sequenceor a reverse complement of a sequence according to SEQ ID NO: 8. In someembodiments, the composition for detecting and/or quantifying total EGFRmRNA further comprises a detectably labeled probe consisting of asequence or a reverse complement of a sequence according to SEQ ID NO: 9or SEQ ID NO: 32.

Additional embodiments provide a composition for detecting and/orquantifying ACTB mRNA. In particular embodiments, the compositioncomprises a primer consisting of a sequence or a reverse complement of asequence according to SEQ ID NO: 13 and a primer consisting of asequence or a reverse complement of a sequence according to SEQ ID NO:14. In some embodiments, the composition for detecting and/orquantifying ACTB mRNA further comprises a detectably labeled probeconsisting of a sequence or a reverse complement of a sequence accordingto SEQ ID NO: 15.

Some embodiments provide a composition for detecting EGFRvIII mRNA andfor quantifying total EGFR mRNA (e.g., in a multiplex, single-tubereaction). In particular embodiments, the composition comprises a primerconsisting of a sequence or a reverse complement of a sequence accordingto SEQ ID NO: 1 or SEQ ID NO: 31, a primer consisting of a sequence or areverse complement of a sequence according to SEQ ID NO: 2, a primerconsisting of a sequence or a reverse complement of a sequence accordingto SEQ ID NO: 7, and a primer consisting of a sequence or a reversecomplement of a sequence according to SEQ ID NO: 8. In some embodimentsof the composition for detecting EGFRvIII mRNA and for quantifying totalEGFR mRNA, the composition further comprises a detectably labeled probeconsisting of a sequence or a reverse complement of a sequence accordingto SEQ ID NO: 3 and a detectably labeled probe consisting of a sequenceor a reverse complement of a sequence according to SEQ ID NO: 9 or SEQID NO: 32. And, in some embodiments of the composition for detectingEGFRvIII mRNA and for quantifying total EGFR mRNA, the compositionfurther comprises primers and probes for quantifying and/or detecting acontrol mRNA such as ACTB. For example, some embodiments provide acomposition for detecting EGFRvIII mRNA and for quantifying total EGFRmRNA comprising a primer consisting of a sequence or a reversecomplement of a sequence according to SEQ ID NO: 13, a primer consistingof a sequence or a reverse complement of a sequence according to SEQ IDNO: 14, and a detectably labeled probe consisting of a sequence or areverse complement of a sequence according to SEQ ID NO: 15.

Particular embodiments related to multiplex detection provide acomposition wherein the detectably labeled probe consisting of asequence or a reverse complement of a sequence according to SEQ ID NO: 3comprises a first distinguishable fluorescent moiety, the detectablylabeled probe consisting of a sequence or a reverse complement of asequence according to SEQ ID NO: 9 or SEQ ID NO: 32 comprises a seconddistinguishable fluorescent moiety, and the detectably labeled probeconsisting of a sequence or a reverse complement of a sequence accordingto SEQ ID NO: 15 comprises a third distinguishable fluorescent moiety,e.g., to detect each probe separately by monitoring a separate detectionchannel in a real-time PCR.

Some embodiments relate to detecting EGFRvIII mRNA and quantifying totalEGFR mRNA in a sample obtained from a subject, e.g., as a cancerdiagnostic. Thus, in some embodiments, compositions further comprise anucleic acid (e.g., RNA or DNA (e.g., cDNA)) prepared from a sampleobtained from a subject.

Some embodiments are related to reaction mixtures for detecting and/orquantifying a nucleic acid (e.g., a RNA or DNA (e.g., cDNA)), e.g., anEGFRvIII nucleic acid ((e.g., an EGFRvIII RNA or DNA (e.g., cDNA)), anEGFR nucleic acid (e.g., an EGFR RNA or DNA (e.g., cDNA), or a controlnucleic acid (e.g., an ACTB, ABL, or G6PD RNA or DNA (e.g., cDNA)).

Accordingly, provided herein are embodiments of a technology related toa reaction mixture for detecting EGFRvIII mRNA. In particularembodiments, the reaction mixture comprises a primer comprising asequence according to SEQ ID NO: 1 or SEQ ID NO: 31 (e.g., a primercomprising a sequence or a reverse complement of a sequence that is atleast 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1 or SEQ IDNO: 31 or that is 100% identical to SEQ ID NO: 1 or SEQ ID NO: 31) and aprimer comprising a sequence according to SEQ ID NO: 2 (e.g., a primercomprising a sequence or a reverse complement of a sequence that is atleast 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 2 or thatis 100% identical to SEQ ID NO: 2). In some embodiments, the reactionmixture for detecting EGFRvIII mRNA further comprises a detectablylabeled probe comprising a sequence according to SEQ ID NO: 3 (e.g., aprobe comprising a sequence or a reverse complement of a sequence thatis at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 3 orthat is 100% identical to SEQ ID NO: 3).

Additional embodiments provide a reaction mixture for detecting and/orquantifying total EGFR mRNA. In particular embodiments, the reactionmixture comprises a primer comprising a sequence according to SEQ ID NO:7 (e.g., a primer comprising a sequence or a reverse complement of asequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical toSEQ ID NO: 7 or that is 100% identical to SEQ ID NO: 7) and a primercomprising a sequence according to SEQ ID NO: 8 (e.g., a primercomprising a sequence or a reverse complement of a sequence that is atleast 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 8 or thatis 100% identical to SEQ ID NO: 8). In some embodiments, the reactionmixture for detecting and/or quantifying total EGFR mRNA furthercomprises a detectably labeled probe comprising a sequence according toSEQ ID NO: 9 or SEQ ID NO: 32 (e.g., a probe comprising a sequence or areverse complement of a sequence that is at least 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to SEQ ID NO: 9 or SEQ ID NO: 32 or that is 100%identical to SEQ ID NO: 9 or SEQ ID NO: 32).

Additional embodiments provide a reaction mixture for detecting and/orquantifying ACTB mRNA. In particular embodiments, the reaction mixturecomprises a primer comprising a sequence according to SEQ ID NO: 13(e.g., a primer comprising a sequence or a reverse complement of asequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical toSEQ ID NO: 13 or that is 100% identical to SEQ ID NO: 13) and a primercomprising a sequence according to SEQ ID NO: 14 (e.g., a primercomprising a sequence or a reverse complement of a sequence that is atleast 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 14 or thatis 100% identical to SEQ ID NO: 14). In some embodiments, the reactionmixture for detecting and/or quantifying ACTB mRNA further comprises adetectably labeled probe comprising a sequence according to SEQ ID NO:15 (e.g., a probe comprising a sequence or a reverse complement of asequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical toSEQ ID NO: 15 or that is 100% identical to SEQ ID NO: 15).

Some embodiments provide a reaction mixture for detecting EGFRvIII mRNAand for quantifying total EGFR mRNA (e.g., in a multiplex, single-tubereaction). In particular embodiments, the reaction mixture comprises aprimer comprising a sequence according to SEQ ID NO: 1 or SEQ ID NO: 31(e.g., a primer comprising a sequence or a reverse complement of asequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical toSEQ ID NO: 1 or SEQ ID NO: 31 or that is 100% identical to SEQ ID NO: 1or SEQ ID NO: 31), a primer comprising a sequence according to SEQ IDNO: 2 (e.g., a primer comprising a sequence or a reverse complement of asequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical toSEQ ID NO: 2 or that is 100% identical to SEQ ID NO: 2), a primercomprising a sequence according to SEQ ID NO: 7 (e.g., a primercomprising a sequence or a reverse complement of a sequence that is atleast 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 7 or thatis 100% identical to SEQ ID NO: 7), and a primer comprising a sequenceaccording to SEQ ID NO: 8 (e.g., a primer comprising a sequence or areverse complement of a sequence that is at least 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to SEQ ID NO: 8 or that is 100% identical to SEQID NO: 8). In some embodiments of the reaction mixture for detectingEGFRvIII mRNA and for quantifying total EGFR mRNA, the reaction mixturefurther comprises a detectably labeled probe comprising a sequenceaccording to SEQ ID NO: 3 (e.g., a probe comprising a sequence or areverse complement of a sequence that is at least 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to SEQ ID NO: 3 or that is 100% identical to SEQID NO: 3) and a detectably labeled probe comprising a sequence accordingto SEQ ID NO: 9 or SEQ ID NO: 32 (e.g., a probe comprising a sequence ora reverse complement of a sequence that is at least 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to SEQ ID NO: 9 or SEQ ID NO: 32 or that is 100%identical to SEQ ID NO: 9 or SEQ ID NO: 32). And, in some embodiments ofthe reaction mixture for detecting EGFRvIII mRNA and for quantifyingtotal EGFR mRNA, the reaction mixture further comprises primers andprobes for quantifying and/or detecting a control mRNA such as ACTB. Forexample, some embodiments provide a reaction mixture for detectingEGFRvIII mRNA and for quantifying total EGFR mRNA comprising a primercomprising a sequence according to SEQ ID NO: 13 (e.g., a primercomprising a sequence or a reverse complement of a sequence that is atleast 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 13 or thatis 100% identical to SEQ ID NO: 13), a primer comprising a sequenceaccording to SEQ ID NO: 14 (e.g., a primer comprising a sequence or areverse complement of a sequence that is at least 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to SEQ ID NO: 14 or that is 100% identical to SEQID NO: 14), and a detectably labeled probe comprising a sequenceaccording to SEQ ID NO: 15 (e.g., a probe comprising a sequence or areverse complement of a sequence that is at least 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to SEQ ID NO: 15 or that is 100% identical to SEQID NO: 15).

Particular embodiments related to multiplex detection provide a reactionmixture wherein the detectably labeled probe comprising a sequenceaccording to SEQ ID NO: 3 (e.g., a probe comprising a sequence or areverse complement of a sequence that is at least 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to SEQ ID NO: 3 or that is 100% identical to SEQID NO: 3) comprises a first distinguishable fluorescent moiety, thedetectably labeled probe comprising a sequence according to SEQ ID NO: 9or SEQ ID NO: 32 (e.g., a probe comprising a sequence or a reversecomplement of a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 9 or SEQ ID NO: 32 or that is 100% identical toSEQ ID NO: 9 or SEQ ID NO: 32) comprises a second distinguishablefluorescent moiety, and the detectably labeled probe comprising asequence according to SEQ ID NO: 15 (e.g., a probe comprising a sequenceor a reverse complement of a sequence that is at least 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% identical to SEQ ID NO: 15 or that is 100% identical toSEQ ID NO: 15) comprises a third distinguishable fluorescent moiety,e.g., to detect each probe separately by monitoring a separate detectionchannel in a real-time PCR.

Some embodiments relate to detecting EGFRvIII mRNA and quantifying totalEGFR mRNA in a sample obtained from a subject, e.g., as a cancerdiagnostic. Thus, in some embodiments, reaction mixtures furthercomprise a nucleic acid (e.g., RNA or DNA (e.g., cDNA)) prepared from asample obtained from a subject.

Further embodiments are related to reaction mixtures for detectingand/or quantifying a nucleic acid (e.g., a RNA or DNA (e.g., cDNA)),e.g., an EGFRvIII nucleic acid ((e.g., an EGFRvIII RNA or DNA (e.g.,cDNA)), an EGFR nucleic acid (e.g., an EGFR RNA or DNA (e.g., cDNA), ora control nucleic acid (e.g., an ACTB, ABL, or G6PD RNA or DNA (e.g.,cDNA)).

For example, in particular embodiments, the reaction mixture comprises aprimer consisting of a sequence or a reverse complement of a sequenceaccording to SEQ ID NO: 1 or SEQ ID NO: 31 and a primer consisting of asequence or a reverse complement of a sequence according to SEQ ID NO:2. In some embodiments, the reaction mixture for detecting EGFRvIII mRNAfurther comprises a detectably labeled probe consisting of a sequence ora reverse complement of a sequence according to SEQ ID NO: 3.

Additional embodiments provide a reaction mixture for detecting and/orquantifying total EGFR mRNA. In particular embodiments, the reactionmixture comprises a primer consisting of a sequence or a reversecomplement of a sequence according to SEQ ID NO: 7 and a primerconsisting of a sequence or a reverse complement of a sequence accordingto SEQ ID NO: 8. In some embodiments, the reaction mixture for detectingand/or quantifying total EGFR mRNA further comprises a detectablylabeled probe consisting of a sequence or a reverse complement of asequence according to SEQ ID NO: 9 or SEQ ID NO: 32.

Additional embodiments provide a reaction mixture for detecting and/orquantifying ACTB mRNA. In particular embodiments, the reaction mixturecomprises a primer consisting of a sequence or a reverse complement of asequence according to SEQ ID NO: 13 and a primer consisting of asequence or a reverse complement of a sequence according to SEQ ID NO:14. In some embodiments, the reaction mixture for detecting and/orquantifying ACTB mRNA further comprises a detectably labeled probeconsisting of a sequence or a reverse complement of a sequence accordingto SEQ ID NO: 15.

Some embodiments provide a reaction mixture for detecting EGFRvIII mRNAand for quantifying total EGFR mRNA (e.g., in a multiplex, single-tubereaction). In particular embodiments, the reaction mixture comprises aprimer consisting of a sequence or a reverse complement of a sequenceaccording to SEQ ID NO: 1 or SEQ ID NO: 31, a primer consisting of asequence or a reverse complement of a sequence according to SEQ ID NO:2, a primer consisting of a sequence or a reverse complement of asequence according to SEQ ID NO: 7, and a primer consisting of asequence or a reverse complement of a sequence according to SEQ ID NO:8. In some embodiments of the reaction mixture for detecting EGFRvIIImRNA and for quantifying total EGFR mRNA, the reaction mixture furthercomprises a detectably labeled probe consisting of a sequence or areverse complement of a sequence according to SEQ ID NO: 3 and adetectably labeled probe consisting of a sequence or a reversecomplement of a sequence according to SEQ ID NO: 9 or SEQ ID NO: 32.And, in some embodiments of the reaction mixture for detecting EGFRvIIImRNA and for quantifying total EGFR mRNA, the reaction mixture furthercomprises primers and probes for quantifying and/or detecting a controlmRNA such as ACTB. For example, some embodiments provide a reactionmixture for detecting EGFRvIII mRNA and for quantifying total EGFR mRNAcomprising a primer consisting of a sequence or a reverse complement ofa sequence according to SEQ ID NO: 13, a primer consisting of a sequenceor a reverse complement of a sequence according to SEQ ID NO: 14, and adetectably labeled probe consisting of a sequence or a reversecomplement of a sequence according to SEQ ID NO: 15.

Particular embodiments related to multiplex detection provide a reactionmixture wherein the detectably labeled probe consisting of a sequence ora reverse complement of a sequence according to SEQ ID NO: 3 comprises afirst distinguishable fluorescent moiety, the detectably labeled probeconsisting of a sequence or a reverse complement of a sequence accordingto SEQ ID NO: 9 or SEQ ID NO: 32 comprises a second distinguishablefluorescent moiety, and the detectably labeled probe consisting of asequence or a reverse complement of a sequence according to SEQ ID NO:15 comprises a third distinguishable fluorescent moiety, e.g., to detecteach probe separately by monitoring a separate detection channel in areal-time PCR.

Some embodiments relate to detecting EGFRvIII mRNA and quantifying totalEGFR mRNA in a sample obtained from a subject, e.g., as a cancerdiagnostic. Thus, in some embodiments, reaction mixtures furthercomprise a nucleic acid (e.g., RNA or DNA (e.g., cDNA)) prepared from asample obtained from a subject.

Further embodiments provide a method for detecting EGFRvIII expressionin a sample. In particular embodiments, the method comprises mixing apatient sample with a composition comprising a primer comprising asequence according to SEQ ID NO: 1 or SEQ ID NO: 31 (e.g., a primercomprising a sequence or a reverse complement of a sequence that is atleast 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1 or SEQ IDNO: 31 or that is 100% identical to SEQ ID NO: 1 or SEQ ID NO: 31), aprimer comprising a sequence according to SEQ ID NO: 2 (e.g., a primercomprising a sequence or a reverse complement of a sequence that is atleast 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 2 or thatis 100% identical to SEQ ID NO: 2), and a detectably labeled probecomprising a sequence according to SEQ ID NO: 3 (e.g., a probecomprising a sequence or a reverse complement of a sequence that is atleast 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 3 or thatis 100% identical to SEQ ID NO: 3) to provide a RT-PCR reaction mixture;reverse transcribing EGFRvIII mRNA to provide an EGFRvIII cDNA;amplifying the EGFRvIII cDNA to provide an EGFRvIII amplicon; anddetecting the EGFRvIII amplicon, wherein detecting the EGFRvIII ampliconindicates the presence of EGFRvIII expression in the sample.

In some embodiments, the technology provides a method for quantifyingand/or detecting total EGFR expression in a sample. In particularembodiments, the method comprises mixing a patient sample with acomposition comprising a primer comprising a sequence according to SEQID NO: 7 (e.g., a primer comprising a sequence or a reverse complementof a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identicalto SEQ ID NO: 7 or that is 100% identical to SEQ ID NO: 7), a primercomprising a sequence according to SEQ ID NO: 8 (e.g., a primercomprising a sequence or a reverse complement of a sequence that is atleast 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 8 or thatis 100% identical to SEQ ID NO: 8), and a detectably labeled probecomprising a sequence according to SEQ ID NO: 9 or SEQ ID NO: 32 (e.g.,a probe comprising a sequence or a reverse complement of a sequence thatis at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 9 orSEQ ID NO: 32 or that is 100% identical to SEQ ID NO: 9 or SEQ ID NO:32) to provide a RT-PCR reaction mixture; reverse transcribing totalEGFR mRNA to provide total EGFR cDNA; amplifying the total EGFR cDNA toprovide a total EGFR amplicon; and detecting or quantifying the totalEGFR amplicon, wherein detecting or quantifying the total EGFR ampliconquantifies and/or detects total EGFR expression in the sample.

In some embodiments, the reverse transcribing, amplifying, and detectingare performed using one or both of RT-PCR or real-time PCR. For example,in some embodiments the detecting comprises determination of a Ct value.

Further embodiments provide a method for detecting EGFRvIII expressionin a sample relative to a control. For example, in some embodiments themethod comprises mixing a patient sample with a composition comprising aprimer comprising a sequence according to SEQ ID NO: 1 or SEQ ID NO: 31(e.g., a primer comprising a sequence or a reverse complement of asequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical toSEQ ID NO: 1 or SEQ ID NO: 31 or that is 100% identical to SEQ ID NO: 1or SEQ ID NO: 31), a primer comprising a sequence according to SEQ IDNO: 2 (e.g., a primer comprising a sequence or a reverse complement of asequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical toSEQ ID NO: 2 or that is 100% identical to SEQ ID NO: 2), a primercomprising a sequence according to SEQ ID NO: 13 (e.g., a primercomprising a sequence or a reverse complement of a sequence that is atleast 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 13 or thatis 100% identical to SEQ ID NO: 13), a primer comprising a sequenceaccording to SEQ ID NO: 14 (e.g., a primer comprising a sequence or areverse complement of a sequence that is at least 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to SEQ ID NO: 14 or that is 100% identical to SEQID NO: 14), a detectably labeled probe comprising a sequence accordingto SEQ ID NO: 3 (e.g., a probe comprising a sequence or a reversecomplement of a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 3 or that is 100% identical to SEQ ID NO: 3),and a detectably labeled probe comprising a sequence according to SEQ IDNO: 15 (e.g., a probe comprising a sequence or a reverse complement of asequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical toSEQ ID NO: 15 or that is 100% identical to SEQ ID NO: 15) to provide aRT-PCR reaction mixture; reverse transcribing EGFRvIII mRNA to provide aEGFRvIII cDNA; reverse transcribing ACTB mRNA to provide an ACTB cDNA;amplifying the EGFRvIII cDNA to provide an EGFRvIII amplicon and anEGFRvIII Ct value; amplifying the ACTB cDNA to provide an ACTB ampliconand an ACTB Ct value; and comparing the EGFRvIII Ct value and the ACTBCt value to provide a dCt value, wherein the dCt value indicates thepresence or absence of EGFRvIII expression in the sample. In someembodiments, the comparing comprises subtracting one Ct value from theother Ct value.

Some embodiments provide a method for quantifying total EGFR expressionin a sample relative to a control. For example, in some embodiments themethod comprises mixing a patient sample with a composition comprising aprimer comprising a sequence according to SEQ ID NO: 7 (e.g., a primercomprising a sequence or a reverse complement of a sequence that is atleast 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 7 or thatis 100% identical to SEQ ID NO: 7), a primer comprising a sequenceaccording to SEQ ID NO: 8 (e.g., a primer comprising a sequence or areverse complement of a sequence that is at least 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to SEQ ID NO: 8 or that is 100% identical to SEQID NO: 8), a primer comprising a sequence according to SEQ ID NO: 13(e.g., a primer comprising a sequence or a reverse complement of asequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical toSEQ ID NO: 13 or that is 100% identical to SEQ ID NO: 13), a primercomprising a sequence according to SEQ ID NO: 14 (e.g., a primercomprising a sequence or a reverse complement of a sequence that is atleast 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 14 or thatis 100% identical to SEQ ID NO: 14), a detectably labeled probecomprising a sequence according to SEQ ID NO: 9 or SEQ ID NO: 32 (e.g.,a probe comprising a sequence or a reverse complement of a sequence thatis at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 9 orSEQ ID NO: 32 or that is 100% identical to SEQ ID NO: 9 or SEQ ID NO:32), and a detectably labeled probe comprising a sequence according toSEQ ID NO: 15 (e.g., a probe comprising a sequence or a reversecomplement of a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 15 or that is 100% identical to SEQ ID NO: 15)to provide a RT-PCR reaction mixture; reverse transcribing total EGFRmRNA to provide total EGFR cDNA; reverse transcribing ACTB mRNA toprovide an ACTB cDNA; amplifying the total EGFR cDNA to provide a totalEGFR amplicon and a total EGFR Ct value; amplifying the ACTB cDNA toprovide an ACTB amplicon and an ACTB Ct value; and comparing the totalEGFR Ct value and the ACTB Ct value to provide a dCt value, wherein thedCt value is used to quantify the total EGFR expression in the samplerelative to a control. In some embodiments, the comparing comprisessubtracting one Ct value from the other Ct value.

Related embodiments provide a method of treating a subject. Inparticular embodiments, the subject has or is at risk of having acancer. Some embodiments provide a method comprising contacting abiological sample obtained from a subject with a primer comprising asequence according to SEQ ID NO: 1 or SEQ ID NO: 31 (e.g., a primercomprising a sequence or a reverse complement of a sequence that is atleast 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1 or SEQ IDNO: 31 or that is 100% identical to SEQ ID NO: 1 or SEQ ID NO: 31), aprimer comprising a sequence according to SEQ ID NO: 2 (e.g., a primercomprising a sequence or a reverse complement of a sequence that is atleast 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 2 or thatis 100% identical to SEQ ID NO: 2), and a detectably labeled probecomprising a sequence according to SEQ ID NO: 3 (e.g., a probecomprising a sequence or a reverse complement of a sequence that is atleast 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 3 or thatis 100% identical to SEQ ID NO: 3); and/or a primer comprising asequence according to SEQ ID NO: 7 (e.g., a primer comprising a sequenceor a reverse complement of a sequence that is at least 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% identical to SEQ ID NO: 7 or that is 100% identical toSEQ ID NO: 7), a primer comprising a sequence according to SEQ ID NO: 8(e.g., a primer comprising a sequence or a reverse complement of asequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical toSEQ ID NO: 8 or that is 100% identical to SEQ ID NO: 8), and adetectably labeled probe comprising a sequence according to SEQ ID NO: 9or SEQ ID NO: 32 (e.g., a probe comprising a sequence or a reversecomplement of a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 9 or SEQ ID NO: 32 or that is 100% identical toSEQ ID NO: 9 or SEQ ID NO: 32); detecting EGFRvIII expression and/orEGFR overexpression in a biological sample obtained from a subject; andadministering an anti-EGFR therapeutic agent to the subject (e.g., ananti-EGFR therapeutic agent that is a monoclonal antibody such as, e.g.,ABT-806 or ABT-414) (see, e.g., Gan et al., J Clin Oncol 31, 2013(suppl; abstr 2520); NIH clinical trials identifier NCT01255657; Gan etal., J Clin Oncol 32:5s, 2014 (suppl; abstr 2021); NIH clinical trialsidentifier NCT01800695; Phillips et al., Mol Cancer Ther 2013; 12(11Suppl):A250).

Further embodiments provide a method for detecting EGFRvIII expressionin a sample. In particular embodiments, the method comprises mixing apatient sample with a composition comprising a primer consisting of asequence or a reverse complement of a sequence according to SEQ ID NO: 1or SEQ ID NO: 31, a primer consisting of a sequence or a reversecomplement of a sequence according to SEQ ID NO: 2, and a detectablylabeled probe consisting of a sequence or a reverse complement of asequence according to SEQ ID NO: 3 to provide a RT-PCR reaction mixture;reverse transcribing EGFRvIII mRNA to provide an EGFRvIII cDNA;amplifying the EGFRvIII cDNA to provide an EGFRvIII amplicon; anddetecting the EGFRvIII amplicon, wherein detecting the EGFRvIII ampliconindicates the presence of EGFRvIII expression in the sample.

In some embodiments, the technology provides a method for quantifyingand/or detecting total EGFR expression in a sample. In particularembodiments, the method comprises mixing a patient sample with acomposition comprising a primer consisting of a sequence or a reversecomplement of a sequence according to SEQ ID NO: 7, a primer consistingof a sequence or a reverse complement of a sequence according to SEQ IDNO: 8, and a detectably labeled probe consisting of a sequence or areverse complement of a sequence according to SEQ ID NO: 9 or SEQ ID NO:32 to provide a RT-PCR reaction mixture; reverse transcribing total EGFRmRNA to provide total EGFR cDNA; amplifying the total EGFR cDNA toprovide a total EGFR amplicon; and detecting or quantifying the totalEGFR amplicon, wherein detecting or quantifying the total EGFR ampliconquantifies and/or detects total EGFR expression in the sample.

In some embodiments, the reverse transcribing, amplifying, and detectingare performed using one or both of RT-PCR or real-time PCR. For example,in some embodiments the detecting comprises determination of a Ct value.

Further embodiments provide a method for detecting EGFRvIII expressionin a sample relative to a control. For example, in some embodiments themethod comprises mixing a patient sample with a composition comprising aprimer consisting of a sequence or a reverse complement of a sequenceaccording to SEQ ID NO: 1 or SEQ ID NO: 31, a primer consisting of asequence or a reverse complement of a sequence according to SEQ ID NO:2, a primer consisting of a sequence or a reverse complement of asequence according to SEQ ID NO: 13, a primer consisting of a sequenceor a reverse complement of a sequence according to SEQ ID NO: 14, adetectably labeled probe consisting of a sequence or a reversecomplement of a sequence according to SEQ ID NO: 3, and a detectablylabeled probe consisting of a sequence or a reverse complement of asequence according to SEQ ID NO: 15 to provide a RT-PCR reactionmixture; reverse transcribing EGFRvIII mRNA to provide an EGFRvIII cDNA;reverse transcribing ACTB mRNA to provide an ACTB cDNA; amplifying theEGFRvIII cDNA to provide an EGFRvIII amplicon and an EGFRvIII Ct value;amplifying the ACTB cDNA to provide an ACTB amplicon and an ACTB Ctvalue; and comparing the EGFRvIII Ct value and the ACTB Ct value toprovide a dCt value, wherein the dCt value indicates the presence orabsence of EGFRvIII expression in the sample. In some embodiments, thecomparing comprises subtracting one Ct value from the other Ct value.

Some embodiments provide a method for quantifying total EGFR expressionin a sample relative to a control. For example, in some embodiments themethod comprises mixing a patient sample with a composition comprising aprimer consisting of a sequence or a reverse complement of a sequenceaccording to SEQ ID NO: 7, a primer consisting of a sequence or areverse complement of a sequence according to SEQ ID NO: 8, a primerconsisting of a sequence or a reverse complement of a sequence accordingto SEQ ID NO: 13, a primer consisting of a sequence or a reversecomplement of a sequence according to SEQ ID NO: 14, a detectablylabeled probe consisting of a sequence or a reverse complement of asequence according to SEQ ID NO: 9 or SEQ ID NO: 32, and a detectablylabeled probe consisting of a sequence or a reverse complement of asequence according to SEQ ID NO: 15 to provide a RT-PCR reactionmixture; reverse transcribing total EGFR mRNA to provide total EGFRcDNA; reverse transcribing ACTB mRNA to provide an ACTB cDNA; amplifyingthe total EGFR cDNA to provide a total EGFR amplicon and a total EGFR Ctvalue; amplifying the ACTB cDNA to provide an ACTB amplicon and an ACTBCt value; and comparing the total EGFR Ct value and the ACTB Ct value toprovide a dCt value, wherein the dCt value is used to quantify the totalEGFR expression in the sample relative to a control. In someembodiments, the comparing comprises subtracting one Ct value from theother Ct value.

Related embodiments provide a method of treating a subject. Inparticular embodiments, the subject has cancer or is at risk of having acancer. Some embodiments provide a method comprising contacting abiological sample obtained from a subject with a primer consisting of asequence or a reverse complement of a sequence according to SEQ ID NO: 1or SEQ ID NO: 31, a primer consisting of a sequence or a reversecomplement of a sequence according to SEQ ID NO: 2, and a detectablylabeled probe consisting of a sequence or a reverse complement of asequence according to SEQ ID NO: 3; and/or a primer consisting of asequence or a reverse complement of a sequence according to SEQ ID NO:7, a primer consisting of a sequence or a reverse complement of asequence according to SEQ ID NO: 8, and a detectably labeled probeconsisting of a sequence or a reverse complement of a sequence accordingto SEQ ID NO: 9 or SEQ ID NO: 32; detecting EGFRvIII expression and/orEGFR overexpression in a biological sample obtained from a subject; andadministering an anti-EGFR therapeutic agent to the subject (e.g., ananti-EGFR therapeutic agent that is a monoclonal antibody such as, e.g.,ABT-806 or ABT-414).

Further embodiments are related to having EGFRvIII expression tested ina sample by another (e.g., another person) according to the technologyprovided herein (e.g., a physician, nurse, or other health-care workerordering, requesting, and/or instructing a laboratory worker or otherperson to test a sample for EGFRvIII expression). In particularembodiments, the method comprises having EGFRvIII expression tested in asample by ordering, requesting, and/or instructing another to test asample for EGFRvIII expression according to a method comprising mixing apatient sample with a composition comprising a primer comprising asequence according to SEQ ID NO: 1 or SEQ ID NO: 31 (e.g., a primercomprising a sequence or a reverse complement of a sequence that is atleast 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1 or SEQ IDNO: 31 or that is 100% identical to SEQ ID NO: 1 or SEQ ID NO: 31), aprimer comprising a sequence according to SEQ ID NO: 2 (e.g., a primercomprising a sequence or a reverse complement of a sequence that is atleast 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 2 or thatis 100% identical to SEQ ID NO: 2), and a detectably labeled probecomprising a sequence according to SEQ ID NO: 3 (e.g., a probecomprising a sequence or a reverse complement of a sequence that is atleast 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 3 or thatis 100% identical to SEQ ID NO: 3) to provide a RT-PCR reaction mixture;reverse transcribing EGFRvIII mRNA to provide an EGFRvIII cDNA;amplifying the EGFRvIII cDNA to provide an EGFRvIII amplicon; anddetecting the EGFRvIII amplicon, wherein detecting the EGFRvIII ampliconindicates the presence of EGFRvIII expression in the sample. In someembodiments, the methods comprise receiving a result indicating thepresence of EGFRvIII expression in the sample

In some embodiments, the technology relates to having total EGFRexpression quantified in a sample by another according to the technologyprovided herein (e.g., a physician, nurse, or other health-care workerordering, requesting, and/or instructing a laboratory worker or otherperson to quantify total EGFR expression in a sample). In particularembodiments, the method comprises having total EGFR expressionquantified in a sample by ordering, requesting, and/or instructinganother to quantify total EGFR expression in a sample by a methodcomprising mixing a patient sample with a composition comprising aprimer comprising a sequence according to SEQ ID NO: 7 (e.g., a primercomprising a sequence or a reverse complement of a sequence that is atleast 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 7 or thatis 100% identical to SEQ ID NO: 7), a primer comprising a sequenceaccording to SEQ ID NO: 8 (e.g., a primer comprising a sequence or areverse complement of a sequence that is at least 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to SEQ ID NO: 8 or that is 100% identical to SEQID NO: 8), and a detectably labeled probe comprising a sequenceaccording to SEQ ID NO: 9 or SEQ ID NO: 32 (e.g., a probe comprising asequence or a reverse complement of a sequence that is at least 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 9 or SEQ ID NO: 32 orthat is 100% identical to SEQ ID NO: 9 or SEQ ID NO: 32) to provide aRT-PCR reaction mixture; reverse transcribing total EGFR mRNA to providetotal EGFR cDNA; amplifying the total EGFR cDNA to provide a total EGFRamplicon; and detecting or quantifying the total EGFR amplicon, whereindetecting or quantifying the total EGFR amplicon quantifies and/ordetects total EGFR expression in the sample. In some embodiments, themethods comprise receiving a result comprising the presences, absence,and/or quantity of total EGFR expression in the sample.

In some embodiments, the reverse transcribing, amplifying, and detectingare performed by another using one or both of RT-PCR or real-time PCR.For example, in some embodiments the detecting performed by anothercomprises determination of a Ct value. In some embodiments, the methodscomprise receiving a Ct value (e.g., relating to the presence, absence,and/or quantity of EGFRvIII and/or total EGFR expression (e.g., relativeto an internal control (e.g., ACTB, ABL, and/or G6PD))).

Further embodiments are related to having EGFRvIII expression tested ina sample by another and relating EGFRvIII expression to a control (e.g.,a physician, nurse, or other health-care worker ordering, requesting,and/or instructing a laboratory worker or other person to test a samplefor EGFRvIII expression relative to a control). For example, in someembodiments the method comprises having EGFRvIII expression tested in asample relative to a control by ordering, requesting, and/or instructinganother to test a sample for EGFRvIII expression relative to a controlby a method comprising mixing a patient sample with a compositioncomprising a primer comprising a sequence according to SEQ ID NO: 1 orSEQ ID NO: 31 (e.g., a primer comprising a sequence or a reversecomplement of a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 1 or SEQ ID NO: 31 or that is 100% identical toSEQ ID NO: 1 or SEQ ID NO: 31), a primer comprising a sequence accordingto SEQ ID NO: 2 (e.g., a primer comprising a sequence or a reversecomplement of a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 2 or that is 100% identical to SEQ ID NO: 2), aprimer comprising a sequence according to SEQ ID NO: 13 (e.g., a primercomprising a sequence or a reverse complement of a sequence that is atleast 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 13 or thatis 100% identical to SEQ ID NO: 13), a primer comprising a sequenceaccording to SEQ ID NO: 14 (e.g., a primer comprising a sequence or areverse complement of a sequence that is at least 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to SEQ ID NO: 14 or that is 100% identical to SEQID NO: 14), a detectably labeled probe comprising a sequence accordingto SEQ ID NO: 3 (e.g., a probe comprising a sequence or a reversecomplement of a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 3 or that is 100% identical to SEQ ID NO: 3),and a detectably labeled probe comprising a sequence according to SEQ IDNO: 15 (e.g., a probe comprising a sequence or a reverse complement of asequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical toSEQ ID NO: 15 or that is 100% identical to SEQ ID NO: 15) to provide aRT-PCR reaction mixture; reverse transcribing EGFRvIII mRNA to provide aEGFRvIII cDNA; reverse transcribing ACTB mRNA to provide an ACTB cDNA;amplifying the EGFRvIII cDNA to provide an EGFRvIII amplicon and anEGFRvIII Ct value; amplifying the ACTB cDNA to provide an ACTB ampliconand an ACTB Ct value; and comparing the EGFRvIII Ct value and the ACTBCt value to provide a dCt value, wherein the dCt value indicates thepresence or absence of EGFRvIII expression in the sample. In someembodiments, the methods comprise receiving a result comprising thepresence or absence of EGFRvIII expression in the sample (e.g., a dCtvalue indicating the presence or absence of EGFRvIII expression in thesample).

Some embodiments are related to having total EGFR expression quantifiedin a sample by another and relating total EGFR expression to a control,e.g., a physician, nurse, or other health-care worker ordering,requesting, and/or instructing a laboratory worker or other person toquantify total EGFR expression relative to a control. For example, insome embodiments the method comprises having total EGFR expressionquantified in a sample relative to a control by ordering, requesting,and/or instructing another to quantify total EGFR expression in a samplerelative to a control by a method comprising mixing a patient samplewith a composition comprising a primer comprising a sequence accordingto SEQ ID NO: 7 (e.g., a primer comprising a sequence or a reversecomplement of a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 7 or that is 100% identical to SEQ ID NO: 7), aprimer comprising a sequence according to SEQ ID NO: 8 (e.g., a primercomprising a sequence or a reverse complement of a sequence that is atleast 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 8 or thatis 100% identical to SEQ ID NO: 8), a primer comprising a sequenceaccording to SEQ ID NO: 13 (e.g., a primer comprising a sequence or areverse complement of a sequence that is at least 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to SEQ ID NO: 13 or that is 100% identical to SEQID NO: 13), a primer comprising a sequence according to SEQ ID NO: 14(e.g., a primer comprising a sequence or a reverse complement of asequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical toSEQ ID NO: 14 or that is 100% identical to SEQ ID NO: 14), a detectablylabeled probe comprising a sequence according to SEQ ID NO: 9 or SEQ IDNO: 32 (e.g., a probe comprising a sequence or a reverse complement of asequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical toSEQ ID NO: 9 or SEQ ID NO: 32 or that is 100% identical to SEQ ID NO: 9or SEQ ID NO: 32), and a detectably labeled probe comprising a sequenceaccording to SEQ ID NO: 15 (e.g., a probe comprising a sequence or areverse complement of a sequence that is at least 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to SEQ ID NO: 15 or that is 100% identical to SEQID NO: 15) to provide a RT-PCR reaction mixture; reverse transcribingtotal EGFR mRNA to provide total EGFR cDNA; reverse transcribing ACTBmRNA to provide an ACTB cDNA; amplifying the total EGFR cDNA to providea total EGFR amplicon and a total EGFR Ct value; amplifying the ACTBcDNA to provide an ACTB amplicon and an ACTB Ct value; and comparing thetotal EGFR Ct value and the ACTB Ct value to provide a dCt value,wherein the dCt value is used to quantify the total EGFR expression inthe sample relative to a control. In some embodiments, the methodscomprise receiving a result comprising a value describing total EGFRexpression in the sample (e.g., a dCt value used to quantify the totalEGFR expression in the sample relative to a control).

Related embodiments provide a method of having a subject treated for adisease, e.g., a disease such as a cancer. For example, some embodimentsrelate to, e.g., a physician, nurse, or other health-care workerordering, requesting, and/or instructing another to treat a subject fora disease based on the result of a test to detect EGFRvIII expressionand/or to quantify total EGFR expression. In particular embodiments, thesubject has cancer or has an increased risk of having a cancer. Forinstance, some embodiments are related to a method comprising contactinga biological sample obtained from a subject with a primer comprising asequence according to SEQ ID NO: 1 or SEQ ID NO: 31 (e.g., a primercomprising a sequence or a reverse complement of a sequence that is atleast 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1 or SEQ IDNO: 31 or that is 100% identical to SEQ ID NO: 1 or SEQ ID NO: 31), aprimer comprising a sequence according to SEQ ID NO: 2 (e.g., a primercomprising a sequence or a reverse complement of a sequence that is atleast 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 2 or thatis 100% identical to SEQ ID NO: 2), and a detectably labeled probecomprising a sequence according to SEQ ID NO: 3 (e.g., a probecomprising a sequence or a reverse complement of a sequence that is atleast 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 3 or thatis 100% identical to SEQ ID NO: 3); and/or a primer comprising asequence according to SEQ ID NO: 7 (e.g., a primer comprising a sequenceor a reverse complement of a sequence that is at least 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% identical to SEQ ID NO: 7 or that is 100% identical toSEQ ID NO: 7), a primer comprising a sequence according to SEQ ID NO: 8(e.g., a primer comprising a sequence or a reverse complement of asequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical toSEQ ID NO: 8 or that is 100% identical to SEQ ID NO: 8), and adetectably labeled probe comprising a sequence according to SEQ ID NO: 9or SEQ ID NO: 32 (e.g., a probe comprising a sequence or a reversecomplement of a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 9 or SEQ ID NO: 32 or that is 100% identical toSEQ ID NO: 9 or SEQ ID NO: 32); detecting EGFRvIII expression and/orEGFR overexpression in a biological sample obtained from a subject; andordering, requesting, and/or instructing another to administer ananti-EGFR therapeutic agent to the subject (e.g., an anti-EGFRtherapeutic agent that is a monoclonal antibody such as, e.g., ABT-806or ABT-414).

Some embodiments provide a method of selectively treating a cancer, themethod comprising selecting a subject for treatment with an anti-EGFRagent on the basis of the patient having EGFRvIII expression; andselectively administering the anti-EGFR agent to the subject.Embodiments of selectively treating a cancer comprise detecting thepresence, absence, or quantity of EGFRvIII according to a methodcomprising mixing a patient sample with a composition comprising aprimer comprising a sequence according to SEQ ID NO: 1 or SEQ ID NO: 31(e.g., a primer comprising a sequence or a reverse complement of asequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical toSEQ ID NO: 1 or SEQ ID NO: 31 or that is 100% identical to SEQ ID NO: 1or SEQ ID NO: 31), a primer comprising a sequence according to SEQ IDNO: 2 (e.g., a primer comprising a sequence or a reverse complement of asequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical toSEQ ID NO: 2 or that is 100% identical to SEQ ID NO: 2), and adetectably labeled probe comprising a sequence according to SEQ ID NO: 3(e.g., a probe comprising a sequence or a reverse complement of asequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical toSEQ ID NO: 3 or that is 100% identical to SEQ ID NO: 3) to provide aRT-PCR reaction mixture; reverse transcribing EGFRvIII mRNA to provide aEGFRvIII cDNA; amplifying the EGFRvIII cDNA to provide an EGFRvIIIamplicon; and detecting the EGFRvIII amplicon, wherein detecting theEGFRvIII amplicon indicates the presence of EGFRvIII expression in thesample.

Some embodiments provide a method of selectively treating a cancer, themethod comprising selecting a subject for treatment with an anti-EGFRagent on the basis of the patient having total EGFR overexpression; andselectively administering the anti-EGFR agent to the subject.Embodiments of selectively treating a cancer comprise detecting thepresence, absence, or quantity of total EGFR overexpression according toa method comprising mixing a patient sample with a compositioncomprising a primer comprising a sequence according to SEQ ID NO: 7(e.g., a primer comprising a sequence or a reverse complement of asequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical toSEQ ID NO: 7 or that is 100% identical to SEQ ID NO: 7), a primercomprising a sequence according to SEQ ID NO: 8 (e.g., a primercomprising a sequence or a reverse complement of a sequence that is atleast 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 8 or thatis 100% identical to SEQ ID NO: 8), and a detectably labeled probecomprising a sequence according to SEQ ID NO: 9 or SEQ ID NO: 32 (e.g.,a probe comprising a sequence or a reverse complement of a sequence thatis at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 9 orSEQ ID NO: 32 or that is 100% identical to SEQ ID NO: 9 or SEQ ID NO:32) to provide a RT-PCR reaction mixture; reverse transcribing totalEGFR mRNA to provide total EGFR cDNA; amplifying the total EGFR cDNA toprovide a total EGFR amplicon; and detecting or quantifying the totalEGFR amplicon, wherein detecting or quantifying the total EGFR ampliconquantifies and/or detects total EGFR expression in the sample.

Further embodiments provide a method for detecting EGFRvIII expressionin a sample and reporting an EGFRvIII expression result and/orrecommending a treatment (e.g., an anti-EGFR treatment). In particularembodiments, the method comprises mixing a patient sample with acomposition comprising a primer comprising a sequence according to SEQID NO: 1 or SEQ ID NO: 31 (e.g., a primer comprising a sequence or areverse complement of a sequence that is at least 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to SEQ ID NO: 1 or SEQ ID NO: 31 or that is 100%identical to SEQ ID NO: 1 or SEQ ID NO: 31), a primer comprising asequence according to SEQ ID NO: 2 (e.g., a primer comprising a sequenceor a reverse complement of a sequence that is at least 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% identical to SEQ ID NO: 2 or that is 100% identical toSEQ ID NO: 2), and a detectably labeled probe comprising a sequenceaccording to SEQ ID NO: 3 (e.g., a probe comprising a sequence or areverse complement of a sequence that is at least 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to SEQ ID NO: 3 or that is 100% identical to SEQID NO: 3) to provide a RT-PCR reaction mixture; reverse transcribingEGFRvIII mRNA to provide an EGFRvIII cDNA; amplifying the EGFRvIII cDNAto provide an EGFRvIII amplicon; detecting the EGFRvIII amplicon,wherein detecting the EGFRvIII amplicon indicates the presence ofEGFRvIII expression in the sample; and reporting the presence ofEGFRvIII expression in the sample and/or recommending a treatment basedon the presence of EGFRvIII in the sample.

In some embodiments, the technology provides a method for quantifyingand/or detecting total EGFR expression in a sample and reporting a totalEGFR expression result or quantity and/or recommending a treatment(e.g., an anti-EGFR treatment). In particular embodiments, the methodcomprises mixing a patient sample with a composition comprising a primercomprising a sequence according to SEQ ID NO: 7 (e.g., a primercomprising a sequence or a reverse complement of a sequence that is atleast 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 7 or thatis 100% identical to SEQ ID NO: 7), a primer comprising a sequenceaccording to SEQ ID NO: 8 (e.g., a primer comprising a sequence or areverse complement of a sequence that is at least 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to SEQ ID NO: 8 or that is 100% identical to SEQID NO: 8), and a detectably labeled probe comprising a sequenceaccording to SEQ ID NO: 9 or SEQ ID NO: 32 (e.g., a probe comprising asequence or a reverse complement of a sequence that is at least 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 9 or SEQ ID NO: 32 orthat is 100% identical to SEQ ID NO: 9 or SEQ ID NO: 32) to provide aRT-PCR reaction mixture; reverse transcribing total EGFR mRNA to providetotal EGFR cDNA; amplifying the total EGFR cDNA to provide a total EGFRamplicon; detecting or quantifying the total EGFR amplicon, whereindetecting or quantifying the total EGFR amplicon quantifies and/ordetects total EGFR expression in the sample; and reporting the presenceor quantity of total EGFR expression in the sample and/or recommending atreatment based on the presence or quantity of total EGFR expression.

In some embodiments, the reverse transcribing, amplifying, and detectingare performed using one or both of RT-PCR or real-time PCR. For example,in some embodiments the detecting comprises determining a Ct value andreporting of a Ct value.

Further embodiments provide a method for detecting EGFRvIII expressionin a sample relative to a control and reporting relative EGFRvIIIexpression in a sample or recommending a treatment (e.g., an anti-EGFRtreatment). For example, in some embodiments the method comprises mixinga patient sample with a composition comprising a primer comprising asequence according to SEQ ID NO: 1 or SEQ ID NO: 31 (e.g., a primercomprising a sequence or a reverse complement of a sequence that is atleast 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1 or SEQ IDNO: 31 or that is 100% identical to SEQ ID NO: 1 or SEQ ID NO: 31), aprimer comprising a sequence according to SEQ ID NO: 2 (e.g., a primercomprising a sequence or a reverse complement of a sequence that is atleast 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 2 or thatis 100% identical to SEQ ID NO: 2), a primer comprising a sequenceaccording to SEQ ID NO: 13 (e.g., a primer comprising a sequence or areverse complement of a sequence that is at least 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to SEQ ID NO: 13 or that is 100% identical to SEQID NO: 13), a primer comprising a sequence according to SEQ ID NO: 14(e.g., a primer comprising a sequence or a reverse complement of asequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical toSEQ ID NO: 14 or that is 100% identical to SEQ ID NO: 14), a detectablylabeled probe comprising a sequence according to SEQ ID NO: 3 (e.g., aprobe comprising a sequence or a reverse complement of a sequence thatis at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 3 orthat is 100% identical to SEQ ID NO: 3), and a detectably labeled probecomprising a sequence according to SEQ ID NO: 15 (e.g., a probecomprising a sequence or a reverse complement of a sequence that is atleast 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 15 or thatis 100% identical to SEQ ID NO: 15) to provide a RT-PCR reactionmixture; reverse transcribing EGFRvIII mRNA to provide an EGFRvIII cDNA;reverse transcribing ACTB mRNA to provide an ACTB cDNA; amplifying theEGFRvIII cDNA to provide an EGFRvIII amplicon and an EGFRvIII Ct value;amplifying the ACTB cDNA to provide an ACTB amplicon and an ACTB Ctvalue; comparing the EGFRvIII Ct value and the ACTB Ct value to providea dCt value, wherein the dCt value indicates the presence or absence ofEGFRvIII expression in the sample; and reporting the presence or absenceof EGFRvIII expression in the sample or recommending a treatment basedon the presence or absence of EGFRvIII expression in the sample.

Some embodiments provide a method for quantifying total EGFR expressionin a sample relative to a control and reporting total EGFR expressionrelative to a control or recommending a treatment (e.g., an anti-EGFRtreatment). For example, in some embodiments the method comprises mixinga patient sample with a composition comprising a primer comprising asequence according to SEQ ID NO: 7 (e.g., a primer comprising a sequenceor a reverse complement of a sequence that is at least 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% identical to SEQ ID NO: 7 or that is 100% identical toSEQ ID NO: 7), a primer comprising a sequence according to SEQ ID NO: 8(e.g., a primer comprising a sequence or a reverse complement of asequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical toSEQ ID NO: 8 or that is 100% identical to SEQ ID NO: 8), a primercomprising a sequence according to SEQ ID NO: 13 (e.g., a primercomprising a sequence or a reverse complement of a sequence that is atleast 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 13 or thatis 100% identical to SEQ ID NO: 13), a primer comprising a sequenceaccording to SEQ ID NO: 14 (e.g., a primer comprising a sequence or areverse complement of a sequence that is at least 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to SEQ ID NO: 14 or that is 100% identical to SEQID NO: 14), a detectably labeled probe comprising a sequence accordingto SEQ ID NO: 9 or SEQ ID NO: 32 (e.g., a probe comprising a sequence ora reverse complement of a sequence that is at least 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to SEQ ID NO: 9 or SEQ ID NO: 32 or that is 100%identical to SEQ ID NO: 9 or SEQ ID NO: 32), and a detectably labeledprobe comprising a sequence according to SEQ ID NO: 15 (e.g., a probecomprising a sequence or a reverse complement of a sequence that is atleast 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 15 or thatis 100% identical to SEQ ID NO: 15) to provide a RT-PCR reactionmixture; reverse transcribing total EGFR mRNA to provide total EGFRcDNA; reverse transcribing ACTB mRNA to provide an ACTB cDNA; amplifyingthe total EGFR cDNA to provide a total EGFR amplicon and a total EGFR Ctvalue; amplifying the ACTB cDNA to provide an ACTB amplicon and an ACTBCt value; comparing the total EGFR Ct value and the ACTB Ct value toprovide a dCt value, wherein the dCt value is used to quantify the totalEGFR expression in the sample relative to a control; and reporting thetotal EGFR expression in the sample relative to a control orrecommending a treatment based on the total EGFR expression in thesample relative to a control.

Related embodiments provide a method of recommending a treatment for asubject based on results provided by an embodiment of a method providedherein. In particular embodiments, the subject has a cancer or is atrisk of having a cancer. Some embodiments provide a method comprisingcontacting a biological sample obtained from a subject with a primercomprising a sequence according to SEQ ID NO: 1 or SEQ ID NO: 31 (e.g.,a primer comprising a sequence or a reverse complement of a sequencethat is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1or SEQ ID NO: 31 or that is 100% identical to SEQ ID NO: 1 or SEQ ID NO:31), a primer comprising a sequence according to SEQ ID NO: 2 (e.g., aprimer comprising a sequence or a reverse complement of a sequence thatis at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 2 orthat is 100% identical to SEQ ID NO: 2), and a detectably labeled probecomprising a sequence according to SEQ ID NO: 3 (e.g., a probecomprising a sequence or a reverse complement of a sequence that is atleast 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 3 or thatis 100% identical to SEQ ID NO: 3); and/or a primer comprising asequence according to SEQ ID NO: 7 (e.g., a primer comprising a sequenceor a reverse complement of a sequence that is at least 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% identical to SEQ ID NO: 7 or that is 100% identical toSEQ ID NO: 7), a primer comprising a sequence according to SEQ ID NO: 8(e.g., a primer comprising a sequence or a reverse complement of asequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical toSEQ ID NO: 8 or that is 100% identical to SEQ ID NO: 8), and adetectably labeled probe comprising a sequence according to SEQ ID NO: 9or SEQ ID NO: 32 (e.g., a probe comprising a sequence or a reversecomplement of a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 9 or SEQ ID NO: 32 or that is 100% identical toSEQ ID NO: 9 or SEQ ID NO: 32); detecting EGFRvIII expression and/orEGFR overexpression in a biological sample obtained from a subject; andrecommending a treatment comprising administering an anti-EGFRtherapeutic agent to the subject (e.g., an anti-EGFR therapeutic agentthat is a monoclonal antibody such as, e.g., ABT-806 or ABT-414).

Embodiments also relate to kits. In particular embodiments, a kit fordetecting EGFRvIII expression and/or quantifying or detecting total EGFRexpression comprises a primer comprising a sequence according to SEQ IDNO: 1 or SEQ ID NO: 31 (e.g., a primer comprising a sequence or areverse complement of a sequence that is at least 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to SEQ ID NO: 1 or SEQ ID NO: 31 or that is 100%identical to SEQ ID NO: 1 or SEQ ID NO: 31) and a primer comprising asequence according to SEQ ID NO: 2 (e.g., a primer comprising a sequenceor a reverse complement of a sequence that is at least 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% identical to SEQ ID NO: 2 or that is 100% identical toSEQ ID NO: 2); a detectably labeled probe comprising a sequenceaccording to SEQ ID NO: 3 (e.g., a probe comprising a sequence or areverse complement of a sequence that is at least 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to SEQ ID NO: 3 or that is 100% identical to SEQID NO: 3); a primer comprising a sequence according to SEQ ID NO: 7(e.g., a primer comprising a sequence or a reverse complement of asequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical toSEQ ID NO: 7 or that is 100% identical to SEQ ID NO: 7) and a primercomprising a sequence according to SEQ ID NO: 8 (e.g., a primercomprising a sequence or a reverse complement of a sequence that is atleast 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 8 or thatis 100% identical to SEQ ID NO: 8); and/or a detectably labeled probecomprising a sequence according to SEQ ID NO: 9 or SEQ ID NO: 32 (e.g.,a probe comprising a sequence or a reverse complement of a sequence thatis at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 9 orSEQ ID NO: 32 or that is 100% identical to SEQ ID NO: 9 or SEQ ID NO:32). Some embodiments of kits further comprise a primer comprising asequence according to SEQ ID NO: 13 (e.g., a primer comprising asequence or a reverse complement of a sequence that is at least 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 13 or that is 100%identical to SEQ ID NO: 13), a primer comprising a sequence according toSEQ ID NO: 14 (e.g., a primer comprising a sequence or a reversecomplement of a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 14 or that is 100% identical to SEQ ID NO: 14),and a detectably labeled probe comprising a sequence according to SEQ IDNO: 15 (e.g., a probe comprising a sequence or a reverse complement of asequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical toSEQ ID NO: 15 or that is 100% identical to SEQ ID NO: 15).

In additional embodiments of kits for detecting EGFRvIII expressionand/or quantifying or detecting total EGFR expression, the kits comprisea primer consisting of a sequence or a reverse complement of a sequenceaccording to SEQ ID NO: 1 or SEQ ID NO: 31 and a primer consisting of asequence or a reverse complement of a sequence according to SEQ ID NO:2; a detectably labeled probe consisting of a sequence or a reversecomplement of a sequence according to SEQ ID NO: 3; a primer consistingof a sequence or a reverse complement of a sequence according to SEQ IDNO: 7 and a primer consisting of a sequence or a reverse complement of asequence according to SEQ ID NO: 8; and/or a detectably labeled probeconsisting of a sequence or a reverse complement of a sequence accordingto SEQ ID NO: 9 or SEQ ID NO: 32. Some embodiments of kits furthercomprise a primer consisting of a sequence or a reverse complement of asequence according to SEQ ID NO: 13, a primer consisting of a sequenceor a reverse complement of a sequence according to SEQ ID NO: 14, and adetectably labeled probe consisting of a sequence or a reversecomplement of a sequence according to SEQ ID NO: 15.

In some embodiments, a computer-based analysis program is used totransform the raw data generated by the detection assay (e.g., datacomprising an EGFRvIII Ct value, a total EGFR Ct value, a EGFRvIII dCtvalue, a total EGFR dCt value; e.g., data comprising an indication ofthe presence, absence, or the amount of EGFRvIII and/or the amount oftotal EGFR expression) into data of predictive value for a physician,clinician, or other. In addition, the present technology comprises insome embodiments the receiving, processing, and transmitting ofinformation to and from laboratories conducting the assays, informationproviders, medical personnel, and subjects. For example, in someembodiments of the present technology, a sample is obtained from asubject and submitted to a profiling service (e.g., a clinical lab at amedical facility, genomic profiling business, etc.) to generate dataand/or a result. The data may be transmitted and/or displayed by anysuitable method. For example, in some embodiments, a profiling servicegenerates a report that is transmitted over a network, printed, and/ordisplayed to the clinician on a computer monitor.

Additional embodiments will be apparent to persons skilled in therelevant art based on the teachings contained herein.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features, aspects, and advantages of the presenttechnology will become better understood with regard to the followingdrawings:

FIG. 1 is a schematic drawing showing primers and probes for detectingand/or quantifying total EGFR mRNA and EGFRvIII mRNA. The locations ofprimers and probes are indicated above the EGFR mRNA and the EGFRvIIImRNA. Exons within non-rearranged EGFR (wtEGFR) mRNA and EGFRvIII mRNAare depicted as numbered boxes. EGFR exons 2-7 (shaded grey, “Regiondeleted in EGFRvIII”) are present in wtEGFR, but are deleted from theEGFRvIII mutant. Exon 1 and exons 8 through 30 are retained in bothwtEGFR and EGFRvIII. As a result of the deletion, exons 1 and 8 arejuxtaposed in EGFRvIII RNA.

FIG. 2A is a schematic drawing showing embodiments of the total EGFR andEGFRvIII primer and probe sequences relative to the cDNA sequencesreverse transcribed from the EGFR and EGFRvIII mRNAs. The cDNA sequenceat the wild-type EGFR exon 29/30 junction is shown in the top portion ofFIG. 2A (“wt”) and the cDNA sequence at the EGFRvIII exon 1/8 junctionis shown in the lower portion of FIG. 2A (“vIII”). The top schematicshows the locations (e.g., binding site, sequence, and/or complementarysequence) of the total EGFR forward primer (e.g., EGwti29_−47 to −25),the total EGFR reverse primer (e.g., EGwti29_+23 to −3), and the totalEGFR probe (e.g., EGi29_−24 to −11pFAM). The bottom schematic shows thelocations (e.g., binding site, sequence, and/or complementary sequence)of the EGFRvIII forward primer (e.g., EGvIIIi1_−58 to −41), the EGFRvIIIreverse primer (e.g., EGFRvIII Rev_D), and the probe (e.g., EGvIIIi1_−39to −21pFAM).

FIG. 2B is a schematic drawing showing embodiments of the total EGFR andEGFRvIII primer and probe sequences relative to the cDNA sequencesreverse transcribed from the EGFR and EGFRvIII mRNAs. The cDNA sequenceat the wild-type EGFR exon 29/30 junction is shown in the top portion ofFIG. 2B (“EGFR”) and the cDNA sequence at the EGFRvIII exon 1/8 junctionis shown in the lower portion of FIG. 2B (“EGFRvIII”). The top schematicshows the locations (e.g., binding site, sequence, and/or complementarysequence) of the total EGFR forward primer (e.g., EGwti29_−47 to −25),the total EGFR reverse primer (e.g., EGwti29_+23 to −3), and the totalEGFR probe (e.g., Total Probe (VIC)). See Table 13. The bottom schematicshows the locations (e.g., binding site, sequence, and/or complementarysequence) of the EGFRvIII forward primer (e.g., EGvIIIi1_−58 to −41),the EGFRvIII reverse primer (e.g., EGFRvIII-A), and the probe (e.g.,EGvIIIi1_−39 to −21pFAM-BHQ1 dT). See Table 13.

FIG. 3 is a schematic drawing showing the beta-actin mRNA and relativelocations (e.g., binding site, sequence, and/or complementary sequence)of primers and probes. Exons comprising beta-actin mRNAs are depicted asnumbered boxes. The location of primers and probes are denoted above themRNA.

FIG. 4 is a schematic drawing showing locations (e.g., binding site,sequence, and/or complementary sequence) of the beta-actin primer andprobe sequences relative to a cDNA (e.g., produced from a beta actinmRNA). The cDNA sequence at the beta-actin exon 1/exon 2 junction isshown.

FIG. 5 is a plot showing a comparison of total EGFR dCt values(beta-actin Ct—EGFR Ct) for GBM FFPE samples. Samples in which EGFRvIIIwas detected are labeled with an asterisk. The EGFRvIII assay indicatedthat EGFRvIII mRNA was detected in 5 of the 21 samples.

FIG. 6 is a plot showing a comparison of EGFRvIII dCt values (beta-actinCt—EGFR Ct) for GBM FFPE samples that had detectable EGFRvIIIexpression. The relative EGFRvIII expression (beta-actin Ct—EGFRvIII Ct)ranged from 2.59 (e.g., “high” expression) to −6.89 (e.g., “low”expression).

FIGS. 7A and 7 b are a series of plots showing detection of total EGFRmRNA relative to the beta-actin control using the total EGFR primersEGwti29_−47 to −25 and EGwti29_+23 to −3 and the total EGFR probeEGi29_−24 to −11pNED. FIG. 7A is a plot showing relative total EGFRexpression (e.g., dCt, e.g., “actin—EGFR”) measured for reactionmixtures comprising the EGFRvIII primers EGvIIIi1_−58 to −41 andEGvIII-A and the EGFRvIII probe EGvIIIi1_−39 to −21pFAM. FIG. 7B is aplot comparing relative total EGFR expression (e.g., dCt, e.g.,“actin—EGFR”) measured for reaction mixtures comprising the EGFRvIIIprimers EGvIIIi1_−58 to −41 and EGvIII-A and the EGFRvIII probeEGvIIIi1_−39 to −21pFAM with the relative total EGFR expression (e.g.,dCt, e.g., “actin—EGFR”) measured for reaction mixtures comprising theEGFRvIII primers EGvIIIi1_−58 to −41 and EGFRvIII Rev_D and the EGFRvIIIprobe EGvIIIi1_−39 to −21pFAM.

FIG. 8A and FIG. 8B are a series of plots showing detection of EGFRvIIImRNA relative to the beta-actin control. FIG. 8A is a plot showingrelative EGFRvIII expression (e.g., dCt, e.g., “actin—EGFRvIII”)measured using the EGFRvIII primers EGvIIIi1_−58 to −41 and EGvIII-A andthe EGFRvIII probe EGvIIIi1_−39 to −21pFAM. FIG. 8B is a plot comparingthe relative EGFRvIII expression (e.g., dCt, e.g., “actin—EGFRvIII”)measured using the EGFRvIII primers EGvIIIi1_−58 to −41 and EGvIII-A andthe EGFRvIII probe EGvIIIi1_−39 to −21pFAM with the relative EGFRvIIIexpression (e.g., dCt, e.g., “actin—EGFR”) measured using the EGFRvIIIprimers EGvIIIi1_−58 to −41 and EGFRvIII Rev_D and the EGFRvIII probeEGvIIIi1_−39 to −21pFAM.

FIG. 9 is a plot showing the linear correlation of the totalconcentration of mRNA isolated from the GBM samples (expressed as log₂)with the mean beta-actin Ct (mean calculated from two independentsamples). The RNA yields for all tested GBM samples ranged from 6.3nanograms per microliter to 237.3 nanograms per microliter.

FIG. 10 is a schematic drawing of the pJW02 plasmid shown in linearform. The plasmid pJW02 comprises the EGFRvIII exon 1/exon 8 junction,the total EGFR exon 29/exon 30 junction that is present in bothwild-type EGFR mRNA and EGFRvIII mRNA, and the beta-actin exon 1/exon 2junction, and thus serves as a positive control for all three targets.

FIG. 11 is a plot showing the linear correlation of mean Ct to theconcentration of EGFRvIII (diamonds), total EGFR (squares), andbeta-actin (triangles) on the pJW02 control plasmid (expressed as log₁₀of concentration in nanograms per reaction). R² correlation coefficientsare above 0.99 for all three targets. Three independent experiments wereperformed at each DNA concentration for each target.

FIG. 12 is a schematic of internal controls showing relative primer andprobe locations (e.g., binding site, sequence, and/or complementarysequence). Only one isoform is shown for each mRNA target, but primerand probe sets target all isoform variants.

FIG. 13 shows the sequence of the mRNA product transcribed from the Homosapiens beta-actin (ACTB) gene. The locations where some embodiments ofdetection primers (arrows) (or their reverse complements) and probes(thick lines) (or their reverse complements) hybridize to the ACTB mRNAor to a cDNA transcribed from the mRNA are indicated. Primer and probesequences are provided in Table 5. Exons are indicated along the rightside of the sequence.

FIG. 14 shows the sequence of the mRNA product transcribed from the Homosapiens c-abl oncogene (ABL). The locations where some embodiments ofdetection primers (arrows) (or their reverse complements) and probes(thick lines) (or their reverse complements) hybridize to the ABL mRNAor to a cDNA transcribed from the mRNA are indicated. Primer and probesequences are provided in Table 5. Exons are indicated along the rightside of the sequence.

FIG. 15 shows the sequence of the mRNA product transcribed from the Homosapiens glucose-6-phosphate dehydrogenase (G6PD) gene. The locationswhere some embodiments of detection primers (arrows) (or their reversecomplements) and probes (thick lines) (or their reverse complements)hybridize to the G6PD mRNA or to a cDNA transcribed from the mRNA areindicated. Primer and probe sequences are provided in Table 5. Exons areindicated along the right side of the sequence.

FIG. 16A and FIG. 16B are a series of plots showing Ct values measuredfor total EGFR, EGFRvIII, and ACTB and the dCt values calculated fortotal EGFR and EGFRvIII relative to ACTB in samples comprising RNAprepared from the EGFRvIII positive control U87vIII. FIG. 16A shows therelationship between the measured total EGFR, EGFRvIII, and ACTB Ctvalues and the total EGFR, EGFRvIII, and ACTB concentrations; FIG. 16Bshows the relationship between the measured total EGFR and EGFRvIII dCtvalues (relative to ACTB) and the total EGFR and EGFRvIIIconcentrations.

FIG. 17A and FIG. 17B are a series of plots showing Ct values measuredfor total EGFR, EGFRvIII, and ABL and the dCt values calculated fortotal EGFR and EGFRvIII relative to ABL in samples comprising RNAprepared from the EGFRvIII positive control U87vIII. FIG. 17A shows therelationship between the measured total EGFR, EGFRvIII, and ABL Ctvalues and the total EGFR, EGFRvIII, and ABL concentrations; FIG. 17Bshows the relationship between the measured total EGFR and EGFRvIII dCtvalues (relative to ABL) and the total EGFR and EGFRvIII concentrations.

FIG. 18A and FIG. 18B are a series of plots showing Ct values measuredfor total EGFR, EGFRvIII, and G6PD and the dCt values calculated fortotal EGFR and EGFRvIII relative to G6PD in samples comprising RNAprepared from the EGFRvIII positive control U87vIII. FIG. 18A shows therelationship between the measured total EGFR, EGFRvIII, and G6PD Ctvalues and the total EGFR, EGFRvIII, and G6PD concentrations; FIG. 18Bshows the relationship between the measured total EGFR and EGFRvIII dCtvalues (relative to G6PD) and the total EGFR and EGFRvIIIconcentrations.

FIG. 19A and FIG. 19B are a series of plots showing Ct values measuredfor total EGFR and ACTB and the dCt values calculated for total EGFRrelative to ACTB in samples comprising RNA prepared from the total EGFRpositive control A549. FIG. 19A shows the relationship between themeasured total EGFR and ACTB Ct values and the total EGFR and ACTBconcentrations; FIG. 19B shows the relationship between the measuredtotal EGFR dCt values (relative to ACTB) and the total EGFRconcentrations.

FIG. 20A and FIG. 20B are a series of plots showing Ct values measuredfor total EGFR and ABL and the dCt values calculated for total EGFRrelative to ABL in samples comprising RNA prepared from the total EGFRpositive control A549. FIG. 20A shows the relationship between themeasured total EGFR and ABL Ct values and the total EGFR and ABLconcentrations; FIG. 20B shows the relationship between the measuredtotal EGFR dCt values (relative to ABL) and the total EGFRconcentrations.

FIG. 21A and FIG. 21B are a series of plots showing Ct values measuredfor total EGFR and G6PD and the dCt values calculated for total EGFRrelative to G6PD in samples comprising RNA prepared from the total EGFRpositive control A549. FIG. 21A shows the relationship between themeasured total EGFR and G6PD Ct values and the total EGFR and G6PDconcentrations; FIG. 21B shows the relationship between the measuredtotal EGFR dCt values (relative to G6PD) and the total EGFRconcentrations.

FIG. 22 is a schematic drawing showing primers and probes for detectingand/or quantifying total EGFR mRNA and EGFRvIII mRNA. Exons comprisingnon-rearranged EGFR mRNA (“EGFR mRNA”, top) and EGFRvIII mRNA (“EGFRvIIImRNA”, bottom) are depicted as numbered boxes. Exons 2-7 (shaded greyand marked with brace labeled “Region deleted in EGFRvIII”) are presentin EGFR, but are deleted from the EGFRvIII deletion variant. Exon 1 andexons 8 through 30 are retained in both EGFR and EGFRvIII and total EGFRprimers and probes amplify both EGFRvIII and EGFR mRNA. As a result ofthe deletion, exons 1 and 8 are juxtaposed in EGFRvIII RNA. Thelocations of primers and probes are indicated above the respectivemRNAs. The EGFRvIII probe is labeled with a “FAM” fluorescent moiety andthe total EGFR probe is labeled with a “VIC” fluorescent moiety. Theprimers associated with each probe are indicated by arrows immediatelyupstream and downstream of the labeled probes.

FIG. 23 is a scatter plot of EGFRvIII Ct from a real-time PCR assay ofRNA dilution panels prepared from FFPE cell pellet of U87MG de2-7 cellline that expresses EGFRvIII, tEGFR, and actin. The averages of datafrom 9 PCR replicates of each dilution panel were plotted; errorbars=SD.

FIG. 24 is a scatter plot of total EGFR Ct from a real-time PCR assay ofRNA dilution panels prepared from FFPE cell pellet of U87MG de2-7 cellline that expresses EGFRvIII, tEGFR, and actin. The averages of datafrom 9 PCR replicates of each dilution panel were plotted; errorbars=SD.

FIG. 25 is a scatter plot of actin Ct from a real-time PCR assay of RNAdilution panels prepared from FFPE cell pellet of U87MG de2-7 cell linethat expresses EGFRvIII, tEGFR, and actin. The averages of data from 9PCR replicates of each dilution panel were plotted; error bars=SD.

It is to be understood that the figures are not necessarily drawn toscale, nor are the objects in the figures necessarily drawn to scale inrelationship to one another. The figures are depictions that areintended to bring clarity and understanding to various embodiments ofapparatuses, systems, and methods disclosed herein. Wherever possible,the same reference numbers will be used throughout the drawings to referto the same or like parts. Moreover, it should be appreciated that thedrawings are not intended to limit the scope of the present teachings inany way.

DETAILED DESCRIPTION

Provided herein is technology relating to detecting cancer andparticularly, but not exclusively, to methods and compositions fordetecting and/or quantifying EGFR and/or EGFRvIII in biological samples.In this detailed description of the various embodiments, for purposes ofexplanation, numerous specific details are set forth to provide athorough understanding of the embodiments disclosed. One skilled in theart will appreciate, however, that these various embodiments may bepracticed with or without these specific details. In other instances,structures and devices are shown in block diagram form. Furthermore, oneskilled in the art can readily appreciate that the specific sequences inwhich methods are presented and performed are illustrative and it iscontemplated that the sequences can be varied and still remain withinthe spirit and scope of the various embodiments disclosed herein. Thesection headings used herein are for organizational purposes only andare not to be construed as limiting the described subject matter in anyway.

All literature and similar materials cited in this application,including but not limited to, patents, patent applications, articles,books, treatises, and internet web pages are expressly incorporatedherein by reference in their entirety for any purpose. Unless definedotherwise, all technical and scientific terms used herein have the samemeaning as is commonly understood by one of ordinary skill in the art towhich the various embodiments described herein belongs. When definitionsof terms in incorporated references appear to differ from thedefinitions provided in the present teachings, the definition providedin the present teachings shall control.

Definitions

To facilitate an understanding of the present technology, a number ofterms and phrases are defined below. Additional definitions are setforth throughout the detailed description.

Throughout the specification and claims, the following terms take themeanings explicitly associated herein, unless the context clearlydictates otherwise. The phrase “in one embodiment” as used herein doesnot necessarily refer to the same embodiment, though it may.Furthermore, the phrase “in another embodiment” as used herein does notnecessarily refer to a different embodiment, although it may. Thus, asdescribed below, various embodiments of the invention may be readilycombined, without departing from the scope or spirit of the invention.

As used herein, a “nucleic acid” or “nucleic acid molecule” generallyrefers to any ribonucleic acid or deoxyribonucleic acid, which may beunmodified or modified DNA or RNA. “Nucleic acids” include, withoutlimitation, single-stranded and double-stranded nucleic acids. As usedherein, the term “nucleic acid” also includes DNA as described abovethat contains one or more modified bases. Thus, DNA with a backbonemodified for stability or for other reasons is a “nucleic acid”. Theterm “nucleic acid” as it is used herein embraces such chemically,enzymatically, or metabolically modified forms of nucleic acids, as wellas the chemical forms of DNA characteristic of viruses and cells,including for example, simple and complex cells.

The terms “oligonucleotide” or “polynucleotide” or “nucleotide” or“nucleic acid” refer to a molecule having two or moredeoxyribonucleotides or ribonucleotides, preferably more than three, andusually more than ten. The exact size will depend on many factors, whichin turn depends on the ultimate function or use of the oligonucleotide.The oligonucleotide may be generated in any manner, including chemicalsynthesis, DNA replication, reverse transcription, or a combinationthereof. Typical deoxyribonucleotides for DNA are thymine, adenine,cytosine, and guanine. Typical ribonucleotides for RNA are uracil,adenine, cytosine, and guanine.

As used herein, the term “antibody” is used in its broadest sense torefer to whole antibodies, monoclonal antibodies (including human,humanized, or chimeric antibodies), polyclonal antibodies, and antibodyfragments that can bind antigen (e.g., Fab′, F′(ab)₂, Fv, single chainantibodies), comprising complementarity determining regions (CDRs) ofthe foregoing as long as they exhibit the desired biological activity.

As used herein, “antibody fragments” comprise a portion of an intactantibody, preferably the antigen binding or variable region of theintact antibody. Examples of antibody fragments include Fab, Fab′,F(ab′)2, and Fv fragments; diabodies; linear antibodies (Zapata et al.,Protein Eng. 8(10): 1057-1062 (1995)); single-chain antibody molecules;and multispecific antibodies formed from antibody fragments.

An antibody that “specifically binds to” or is “specific for” aparticular polypeptide or an epitope on a particular polypeptide is onethat binds to that particular polypeptide or epitope on a particularpolypeptide without substantially binding to any other polypeptide orpolypeptide epitope.

As used herein, the terms “locus” or “region” of a nucleic acid refer toa subregion of a nucleic acid.

The terms “complementary” and “complementarity” refer to nucleotides(e.g., 1 or more nucleotide) or polynucleotides (e.g., a sequence of aplurality nucleotides) related by the base-pairing rules. For example,the sequence 5′-A-G-T-3′ is complementary to the sequence 3′-T-C-A-5′.Complementarity may be “partial,” in which only some of the nucleicacids' bases are matched according to the base pairing rules. Or, theremay be “complete” or “total” complementarity between the nucleic acids.The degree of complementarity between nucleic acid strands effects theefficiency and strength of hybridization between nucleic acid strands.This is of particular importance in amplification reactions and indetection methods that depend upon binding between nucleic acids.

The term “gene” refers to a nucleic acid (e.g., DNA or RNA) sequencethat comprises coding sequences necessary for the production of a RNA orof a polypeptide or its precursor. A functional polypeptide can beencoded by a full length coding sequence or by any portion of the codingsequence as long as the desired activity or functional properties (e.g.,enzymatic activity, ligand binding, signal transduction, etc.) of thepolypeptide are retained. The term “portion” when used in reference to agene refers to fragments of that gene. The fragments may range in sizefrom a few (e.g., 1 to 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10)nucleotides to the entire gene sequence minus one nucleotide. Thus, “anucleotide comprising at least a portion of a gene” may comprisefragments of the gene or the entire gene.

The term “gene” also encompasses the coding regions of a structural geneand includes sequences located adjacent to the coding region on both the5′ and 3′ ends, e.g., for a distance of about 1 kb on either end, suchthat the gene corresponds to the length of the full-length mRNA (e.g.,comprising coding, regulatory, structural and other sequences). Thesequences that are located 5′ of the coding region and that are presenton the mRNA are referred to as 5′ non-translated or untranslatedsequences. The sequences that are located 3′ or downstream of the codingregion and that are present on the mRNA are referred to as 3′non-translated or 3′ untranslated sequences. The term “gene” encompassesboth cDNA and genomic forms of a gene. In some organisms (e.g.,eukaryotes), a genomic form or clone of a gene contains the codingregion interrupted with non-coding sequences termed “introns” or“intervening regions” or “intervening sequences.” Introns are segmentsof a gene that are transcribed into nuclear RNA (hnRNA); introns maycontain regulatory elements such as enhancers. Introns are removed or“spliced out” from the nuclear or primary transcript; introns thereforeare absent in the messenger RNA (mRNA) transcript. The mRNA functionsduring translation to specify the sequence or order of amino acids in anascent polypeptide.

In addition to containing introns, genomic forms of a gene may alsoinclude sequences located on both the 5′ and 3′ ends of the sequencesthat are present on the RNA transcript. These sequences are referred toas “flanking” sequences or regions (these flanking sequences are located5′ or 3′ to the non-translated sequences present on the mRNAtranscript). The 5′ flanking region may contain regulatory sequencessuch as promoters and enhancers that control or influence thetranscription of the gene. The 3′ flanking region may contain sequencesthat direct the termination of transcription, posttranscriptionalcleavage, and polyadenylation.

The term “wild-type” when made in reference to a gene refers to a genethat has the characteristics of a gene isolated from a naturallyoccurring source. The term “wild-type” when made in reference to a geneproduct refers to a gene product that has the characteristics of a geneproduct isolated from a naturally occurring source. The term“naturally-occurring” as applied to an object refers to the fact that anobject can be found in nature. For example, a polypeptide orpolynucleotide sequence that is present in an organism (includingviruses) that can be isolated from a source in nature and which has notbeen intentionally modified by the hand of a person in the laboratory isnaturally-occurring. A wild-type gene is often that gene or allele thatis most frequently observed in a population and is thus arbitrarilydesignated the “normal” or “wild-type” form of the gene (e.g., wild-typeEGFR). In contrast, the term “modified” or “mutant” when made inreference to a gene or to a gene product refers, respectively, to a geneor to a gene product that displays modifications in sequence and/orfunctional properties (e.g., altered characteristics) when compared tothe wild-type gene or gene product (e.g., EGFRvIII). It is noted thatnaturally-occurring mutants can be isolated; these are identified by thefact that they have altered characteristics when compared to thewild-type gene or gene product.

As used herein, “total EGFR” refers to EGFR mRNA in all forms and, inparticular, “total EGFR” refers to all EGFR mRNA that comprises the exon29/exon 30 junction, e.g., EGFR mRNA in both the “wild-type EGFR”(“wtEGFR”) and EGFRvIII forms.

The term “allele” refers to a variation of a gene; the variationsinclude but are not limited to variants and mutants, polymorphic loci,and single nucleotide polymorphic loci, frameshift, and splicemutations. An allele may occur naturally in a population or it mightarise during the lifetime of any particular individual of thepopulation.

Thus, the terms “variant” and “mutant” when used in reference to anucleotide sequence refer to a nucleic acid sequence that differs by oneor more nucleotides from another, usually related, nucleotide acidsequence. A “variation” is a difference between two different nucleotidesequences; typically, one sequence is a reference sequence.

“Amplification” is a special case of nucleic acid replication involvingtemplate specificity. It is to be contrasted with non-specific templatereplication (e.g., replication that is template-dependent but notdependent on a specific template). Template specificity is heredistinguished from fidelity of replication (e.g., synthesis of theproper polynucleotide sequence) and nucleotide (ribonucleotide ordeoxyribonucleotide) specificity. Template specificity is frequentlydescribed in terms of “target” specificity. Target sequences are“targets” in the sense that they are sought to be sorted out from othernucleic acids. Amplification techniques have been designed primarily forthis sorting out.

Amplification of nucleic acids generally refers to the production ofmultiple copies of a polynucleotide, or a portion of the polynucleotide,typically starting from a small amount of the polynucleotide (e.g., asingle polynucleotide molecule, 10 to 100 copies of a polynucleotidemolecule, which may or may not be exactly the same), where theamplification products or amplicons are generally detectable.Amplification of polynucleotides encompasses a variety of chemical andenzymatic processes including polymerase chain reaction (PCR) andreverse transcription-polymerase chain reaction (RT-PCR).

The term “polymerase chain reaction” (“PCR”) refers a method forincreasing the concentration of a segment of a target sequence in amixture of genomic DNA without cloning or purification. The length ofthe amplified segment of the desired target sequence is determined bythe relative positions of the primers with respect to each other, andtherefore, this length is a controllable parameter. Because the desiredamplified segments of the target sequence become the predominantsequences (in terms of concentration) in the mixture, they are said tobe “PCR amplified” and are “PCR products” or “amplicons.”

As used herein, the term “RT-PCR reaction mixture” means a compositioncomprising elements appropriate to perform reversetranscription-polymerase chain reaction including but not limited toprimers having specificity for the sequence of the diagnostic targetRNA; a polymerase (e.g., a thermostable polymerase, e.g., heat activatedthermostable polymerase); dNTPs, and appropriate buffers. In someembodiments, the polymerase comprises a reverse transcriptase activity.In some embodiments, the reverse transcriptase activity and polymeraseactivity are provided by two different enzymes.

As used herein, the term “RT reaction mixture” means a compositioncomprising elements appropriate to synthesize a DNA product from an RNAtemplate, including but not limited to a reverse transcriptase enzyme,nucleic acid primer(s) complementary to the target RNA, dNTPs, and theappropriate buffers, and may further contain detection dyes or probes.

As used herein, the term “PCR reaction mixture” means a compositioncomprising elements appropriate to amplify a DNA template, including butnot limited to nucleic acid primers, thermostable polymerases, dNTPs,and appropriate buffers, and may further contain detection dyes orprobes.

The term “sample template” refers to nucleic acid originating from asample that is analyzed for the presence of “target” (defined below). Incontrast, “background template” is used in reference to nucleic acidother than sample template that may or may not be present in a sample.Background template is most often inadvertent. It may be the result ofcarryover or it may be due to the presence of nucleic acid contaminantssought to be purified away from the sample. For example, nucleic acidsfrom organisms other than those to be detected may be present asbackground in a test sample.

The term “primer” refers to an oligonucleotide, whether occurringnaturally as in a purified restriction digest or produced synthetically,that is capable of acting as a point of initiation of synthesis whenplaced under conditions in which synthesis of a primer extension productthat is complementary to a nucleic acid strand is induced, (e.g., in thepresence of nucleotides and an inducing agent such as a DNA polymeraseand at a suitable temperature and pH). The primer is preferably singlestranded for maximum efficiency in amplification, but may alternativelybe double stranded. If double stranded, the primer is first treated toseparate its strands before being used to prepare extension products.Preferably, the primer is an oligodeoxyribonucleotide. The primer mustbe sufficiently long to prime the synthesis of extension products in thepresence of the inducing agent. The exact lengths of the primers willdepend on many factors, including temperature, source of primer, and theuse of the method. Within the context of reverse transcription, primersare composed of nucleic acids and prime on RNA templates. Within thecontext of PCR, primers are composed of nucleic acids and prime on DNAtemplates.

The term “probe” refers to an oligonucleotide (e.g., a sequence ofnucleotides), whether occurring naturally as in a purified restrictiondigest or produced synthetically, recombinantly, or by PCRamplification, that is capable of hybridizing to another oligonucleotideof interest. A probe may be single-stranded or double-stranded. Probesare useful in the detection, identification, and isolation of particulargene sequences (e.g., a “capture probe”). It is contemplated that anyprobe used in the present invention may, in some embodiments, be“detectably labeled” or “labeled” with any “label” or “reporter moiety,”so that it is detectable in any detection system, including, but notlimited to enzyme (e.g., ELISA, as well as enzyme-based histochemicalassays), fluorescent, radioactive, and luminescent systems. It is notintended that the present invention be limited to any particulardetection system or label.

The term “hybridization” is to be understood as the binding of anoligonucleotide to a complementary sequence in a nucleic acid by anumber of Watson-Crick base pairings to form a duplex structure.

As used herein, the term “neoplasm” refers to “an abnormal mass oftissue, the growth of which exceeds and is uncoordinated with that ofthe normal tissues” See, e.g., Willis R A, “The Spread of Tumors in theHuman Body”, London, Butterworth & Co, 1952.

The term “pre-cancerous” or “pre-neoplastic” and equivalents thereofrefer to any cellular proliferative disorder that is undergoingmalignant transformation.

A “site” of a neoplasm, adenoma, cancer, etc. is the tissue, organ, celltype, anatomical area, body part, etc. in a subject's body where theneoplasm, adenoma, cancer, etc. is located.

As used herein, a “diagnostic” test application includes the detectionor identification of a disease state or condition of a subject,determining the likelihood that a subject will contract a given diseaseor condition, determining the likelihood that a subject with a diseaseor condition will respond to therapy, determining the prognosis of asubject with a disease or condition (or its likely progression orregression), and determining the effect of a treatment on a subject witha disease or condition. For example, a diagnostic can be used fordetecting the presence or likelihood of a subject contracting a neoplasmor the likelihood that such a subject will respond favorably to acompound (e.g., a pharmaceutical, e.g., a drug) or other treatment.

As used herein, the term “treating” includes reducing or alleviating atleast one adverse effect or symptom of a disease or disorder.“Treatment” refers to both therapeutic treatment and prophylactic orpreventative measures, wherein the object is to prevent or slow down(lessen) the targeted pathologic condition or disorder. Those in need oftreatment include those already with the disorder as well as those proneto have the disorder or those in whom the disorder is to be prevented.

The term “isolated” when used in relation to a nucleic acid, as in “anisolated oligonucleotide” refers to a nucleic acid sequence that isidentified and separated from at least one contaminant nucleic acid withwhich it is ordinarily associated in its natural source. Isolatednucleic acid is present in a form or setting that is different from thatin which it is found in nature. In contrast, non-isolated nucleic acids,such as DNA and RNA, are found in the state they exist in nature.Examples of non-isolated nucleic acids include: a given DNA sequence(e.g., a gene) found on the host cell chromosome in proximity toneighboring genes; RNA sequences, such as a specific mRNA sequenceencoding a specific protein, found in the cell as a mixture withnumerous other mRNAs which encode a multitude of proteins. However,isolated nucleic acid encoding a particular protein includes, by way ofexample, such nucleic acid in cells ordinarily expressing the protein,where the nucleic acid is in a chromosomal location different from thatof natural cells, or is otherwise flanked by a different nucleic acidsequence than that found in nature. The isolated nucleic acid oroligonucleotide may be present in single-stranded or double-strandedform. When an isolated nucleic acid or oligonucleotide is to be utilizedto express a protein, the oligonucleotide will contain at a minimum thesense or coding strand (i.e., the oligonucleotide may besingle-stranded), but may contain both the sense and anti-sense strands(i.e., the oligonucleotide may be double-stranded). An isolated nucleicacid may, after isolation from its natural or typical environment, by becombined with other nucleic acids or molecules. For example, an isolatednucleic acid may be present in a host cell in which into which it hasbeen placed, e.g., for heterologous expression.

The term “purified” refers to molecules, either nucleic acid or aminoacid sequences that are removed from their natural environment,isolated, or separated. An “isolated nucleic acid sequence” maytherefore be a purified nucleic acid sequence. “Substantially purified”molecules are at least 60% free, preferably at least 75% free, and morepreferably at least 90% free from other components with which they arenaturally associated. As used herein, the terms “purified” or “topurify” also refer to the removal of contaminants from a sample. Theremoval of contaminating proteins results in an increase in the percentof polypeptide or nucleic acid of interest in the sample. In anotherexample, recombinant polypeptides are expressed in plant, bacterial,yeast, or mammalian host cells and the polypeptides are purified by theremoval of host cell proteins; the percent of recombinant polypeptidesis thereby increased in the sample.

The term “composition comprising” a given nucleotide sequence orpolypeptide sequence refers broadly to any composition containing anucleic acid having the given nucleotide sequence or a polypeptidehaving the given amino acid (e.g., polypeptide) sequence. Thecomposition may comprise other components.

The term “sample” is used in its broadest sense. In one sense it canrefer to an animal cell or tissue. In another sense, it is meant toinclude a specimen or culture obtained from any source, as well asbiological and environmental samples. Biological samples may be obtainedfrom plants or animals (including humans) and encompass fluids, solids,tissues, and gases. Materials obtained from clinical or forensicsettings that contain nucleic acids are also within the intended meaningof the term “sample”. Preferably, the sample is a biological samplederived from an animal, e.g., a human. The term “sample” also includesprocessed samples including preserved, fixed, and/or stabilized samples,such as formalin fixed and paraffin-embedded (FFPE samples) and othersamples that were treated with cross-linking fixatives such asglutaraldehyde. These examples are not to be construed as limiting thesample types applicable to the present technology.

As used herein, the terms “patient” or “subject” refer to organisms tobe subject to various tests provided by the technology. The term“subject” includes animals, preferably mammals, including humans. In apreferred embodiment, the subject is a primate. In an even morepreferred embodiment, the subject is a human.

As used herein, the term “reverse transcription followed by polymerasechain reaction”, or “RT-PCR”, refers to a technique for synthesizing acDNA from RNA and amplifying the cDNA molecule. RT-PCR is useful fordetecting RNA species such as in quantitative analysis of geneexpression and for producing DNA copies of RNA for use in cloning, cDNAlibrary construction, probe synthesis, and signal amplification in insitu hybridizations. The technique comprises two parts: 1) synthesis ofcDNA from RNA by reverse transcription (RT); and 2) amplification of aspecific cDNA by polymerase chain reaction (PCR). Reverse transcriptaseis an RNA dependent DNA polymerase that catalyzes the polymerization ofnucleotides using template RNA or the RNA molecule in an RNA:DNA hybrid.Typically, both the reverse transcription and the PCR reactions of theRT-PCR reaction are performed in a single tube.

The term “multiplex amplification” as used herein means the simultaneousamplification of multiple DNA or RNA target sequences in a singlemixture.

The term “multiplex RT-PCR” as used herein means reverse transcribingthe multiple RNA molecules in a sample that contains a mixture of RNAand DNA molecules to produce a mixture that contains multiple cDNA andDNA molecules, and then simultaneously PCR amplifying particular targetsequences from the multiple cDNA and DNA molecules in a single reactionmixture.

As used herein, the term “cycle threshold” or “Ct” refers to thethreshold in RT-PCR at which the fluorescence generated within areaction well exceeds an established threshold or cutoff (e.g.,“baseline”) level. The cycle threshold refers to the same value as dothe terms “crossing point” (Cp) and “take-off point” (TOP) used bycompeting manufacturers of real-time PCR instruments for reasons ofproduct differentiation, and the term “quantification cycle” (Cq) asproposed by the MIQE Guidelines (Bustin et al., Clinical Chemistry,55:4, pp. 611-622 (2009)). As used herein, the term “dCt”, “delta Ct”,or “ΔCt” refers to the difference in two Ct values, e.g., the Ct valuefor a nucleic acid of interest subtracted from the Ct value for acontrol or the Ct value for a control subtracted from the Ct value for anucleic acid of interest.

As used herein, the terms “reference”, “reference gene”, “referencemarker”, “reference target”, “control”, “control marker”, “controltarget” refer to a reference molecule that controls and/or can be usedto control for potential process interfering factors and/or provides oneor more indications about the sample quality, the effective samplepreparation, and/or assembly of the RT-PCR reaction in the sample. Acontrol may either be co-detected or detected separately from targets.

As used herein, “gene expression” or “expression” refers to the absoluteor relative levels of expression and/or pattern of expression of a gene.The expression of a gene may be measured at the level of DNA, cDNA, RNA,mRNA, or combinations thereof. Expression may be measured in a sample byvarious methods including but not limited to microarray technologies andquantitative and semi-quantitative RT-PCR techniques. Measures ofexpression may be qualitative or quantitative, e.g., expression can beabsent, present, detectable, undetectable, below a detection threshold(or, alternatively, below a limit of detection (LOD)), above a detectionthreshold (or, alternatively, above a limit of detection (LOD)), a valueof zero, a value less than zero, or a value more than zero.

As used herein, the word “presence” or “absence” (or, alternatively,“present or “absent”) is used in a relative sense to describe the amountor level of a particular entity (e.g., a nucleic acid (e.g., an RNA(e.g., a mRNA))). For example, when a nucleic acid is said to be“present” in a test sample, it means the level or amount of this nucleicacid is above a pre-determined threshold; conversely, when a nucleicacid is said to be “absent” in a test sample, it means the level oramount of this nucleic acid is below a pre-determined threshold. Thepre-determined threshold may be the threshold for detectabilityassociated with the particular test used to detect the nucleic acid orany other threshold. When a nucleic acid is “detected” or “detectable”in a sample it is “present” in the sample; when a nucleic acid is “notdetected” or “not detectable” it is “absent” from the sample. Further, asample in which a nucleic acid is “detected” or “detectable” or in whichthe nucleic acid is “present” is a sample that is “positive” for thenucleic acid. A sample in which a nucleic acid is “not detected” or “notdetectable” or in which the nucleic acid is “absent” is a sample that is“negative” for the nucleic acid.

In some embodiments, “detecting” a nucleic acid refers to determining ifthe nucleic acid is “present” or “absent”; in some embodiments,“detecting” further comprises “quantifying” the nucleic acid, e.g.,measuring an amount of the nucleic acid.

As used herein, an “increase” or a “decrease” refers to a detectable(e.g., measured) positive or negative change in the value of a variablerelative to a previously measured value of the variable, relative to apre-established value (e.g., a previously known value, a publishedvalue, etc.), and/or relative to a value of a standard control. Anincrease is a positive change preferably at least 10%, more preferably50%, still more preferably 2-fold, even more preferably at least 5-fold,and most preferably at least 10-fold relative to the previously measuredvalue of the variable, the pre-established value, and/or the value of astandard control. Similarly, a decrease is a negative change preferablyat least 10%, more preferably 50%, still more preferably at least 80%,and most preferably at least 90% of the previously measured value of thevariable, the pre-established value, and/or the value of a standardcontrol. Other terms indicating quantitative changes or differences,such as “more” or “less,” are used herein in the same fashion asdescribed above.

As used herein, a “system” refers to a set of components, real orabstract, comprising a whole and in which each component interacts withor is related to at least one other component within the whole.

In addition, as used herein, the term “or” is an inclusive “or” operatorand is equivalent to the term “and/or” unless the context clearlydictates otherwise. The term “based on” is not exclusive and allows forbeing based on additional factors not described, unless the contextclearly dictates otherwise. In addition, throughout the specification,the meaning of “a”, “an”, and “the” include plural references. Themeaning of “in” includes “in” and “on.”

DESCRIPTION

Although the disclosure herein refers to certain illustratedembodiments, it is to be understood that these embodiments are presentedby way of example and not by way of limitation.

EGFRvIII Amplification Primers and Hybridization Probes

In some embodiments, the technology provided herein relates to detectionand/or quantification of EGFRvIII and/or total EGFR RNA expression.During the development of embodiments of the technology, twooligonucleotide primers (“RPvIII” and “FPvIII”) and one hybridizationprobe (“PBvIII”) were developed that target EGFRvIII RNA. Exemplaryprimer and probe sequences contemplated for use in the technology areprovided in TABLE 1 and TABLE 5; see, e.g., FIG. 1 and FIG. 2. As usedherein, the terms “FPvIII”, “RPvIII”, and “PBvIII” refer to forwardprimers, reverse primers, and hybridization probes targeting EGFRvIIIRNA or EGFRvIII cDNA as described herein and as exemplified by thespecific primers and probes contemplated and described.

These primers and probe find use in the specific amplification (e.g., byRT-PCR) and detection of EGFRvIII RNA (e.g., the primers and probeamplify and detect EGFRvIII RNA and the primers and probe do not amplifyand detect wild-type EGFR RNA). In particular, the reverse primer RPvIIIanneals to a specific sequence spanning the fusion junction of EGFR exon1 and EGFR exon 8 that is present in EGFRvIII but that is not present inwild-type (e.g., non-rearranged) EGFR (see, e.g., FIG. 1). Since theEGFR exon 1/exon 8 junction sequence is specific to EGFRvIII RNA, thisdesign provides that the cDNA generated during amplification by theextension of reverse primer RPvIII is specifically produced fromEGFRvIII RNA and is not produced from wild-type (non-rearranged) EGFRRNA or from genomic DNA. Amplification (e.g., by PCR) of the resultingEGFRvIII cDNA is directed by the reverse primer RPvIII in combinationwith forward primer FPvIII, which is specific to EGFR exon 1 (see, e.g.,FIG. 1 and FIG. 2). The fluorescently labeled probe (PBvIII) targetsEGFR exon 1 sequences between the forward primer FPvIII and reverseprimer RPvIII and thus provides for the detection of theEGFRvIII-specific amplification product (e.g., by real-time PCR).

TABLE 1 EGFRvIII oligonucleotides SEQ ID name description target sitesequence (5′ to 3′) NO RPvIII EGFRvIII reverse fusion junction of EGFR           ACCACATAATTACctttc  1 primer exon 1 and EGFR exon 8 RPvIIIEGFRvIII reverse fusion junction of EGFR CGTGATCTGTCACCACATAATTACctttc31 primer exon 1 and EGFR exon 8 FPvIII EGFRvIII forward EGFR exon 1CTCCTGGCGCTGCTGGCT  2 primer PBvIII EGFRvIII probe EGFR exon 1D-CGCTCTGCCCGGCGAGTCG-Q  3

In TABLE 1, upper case letters in the reverse primer sequence RPvIII(e.g., ACCACATAATTAC (SEQ ID NO: 4)) indicate the bases of the primerthat hybridize to the 5′ end of EGFR exon 8 and lower case letters inthe reverse primer sequence RPvIII (e.g., ctttc (SEQ ID NO: 5)) indicatethe bases of the primer that hybridize to the 3′ end of EGFR exon 1.Further, the probe sequence provided in TABLE 1 is derived from thesense strand of the EGFR gene, but the technology also encompasses useof a probe having a sequence that is complementary to the sequence ofPBvIII provided in TABLE 1 (e.g., CGACTCGCCGGGCAGAGCG (SEQ ID NO: 6)).Embodiments provide that the dye D is any moiety (e.g., a dye) that canfluoresce and allow effective association of the probe to the target.Embodiments provide that the quencher Q in probe PBvIII at the oppositeend from the dye D in probe PBvIII is any moiety that effectivelyquenches the fluorescent dye and allows effective association (e.g.,hybridization) of these probes to their target sequences. In TABLE 1,the dye D is attached to PBvIII at the 5′ end of PBvIII and the quencherQ is attached to PBvIII at the 3′ end of PBvIII; however, the technologyalso includes a probe in which the dye D is attached to the probe at the3′ end of the probe and the quencher Q is attached to the probe at the5′ end of the probe. The probe in TABLE 1 does not limit the technology.For instance, the technology also provides embodiments in which a probecomprises a detectable label at the 5′ end and/or at the 3′ end, e.g., aprobe that comprises a dye D at the 5′ end or at the 3′ end. Someembodiments provide a probe that does not comprise a quencher. In someembodiments, the primers and/or the probe are modified, e.g., to includea variety of binding enhancers such as a minor groove binder (“MGB”) ora pdU or pdC nucleotide.

Total EGFR Amplification Primers and Hybridization Probes

In some embodiments, the technology relates to the amplification anddetection of total EGFR RNA and/or the detection and/or quantificationof total EGFR expression (e.g., comprising both expression ofnon-rearranged wild type EGFR and expression of the EGFRvIII mutant).During the development of embodiments of the technology, twooligonucleotide primers (“RPtot” and “FPtot”) and one hybridizationprobe (“PBtot”) were developed that target total EGFR RNA. Exemplaryprimer and probe sequences contemplated for use in the technology areprovided in TABLE 2; see, e.g., FIG. 1 and FIG. 2. As used herein, theterms “FPtot”, “RPtot”, and “PBtot” refer to forward primers, reverseprimers, and hybridization probes targeting total EGFR RNA or total EGFRcDNA (e.g., comprising RNA and/or cDNA of both the non-rearranged wildtype EGFR and the EGFRvIII mutant) as described herein and asexemplified by the specific primers and probes contemplated anddescribed.

These primers and probe find use in the amplification (e.g., by RT-PCR)and detection of both EGFR RNA and EGFRvIII RNA. In particular, thereverse primer RPtot anneals to a specific sequence spanning the splicejunction of EGFR exon 29 and EGFR exon 30 (see, e.g., FIG. 1 and FIG.2). The EGFR exon 29/exon 30 junction sequence is present in RNAsencoded by both wild type EGFR (non-rearranged) and EGFRvIII (see, e.g.,FIG. 1 and FIG. 2), but not in the genomic DNA. Therefore, reverseprimer RPtot provides an amplification product (e.g., a cDNA) fromwild-type EGFR RNA and EGFRvIII RNA, but does not provide anamplification product from wild-type EGFR genomic DNA or from EGFRvIIIgenomic DNA. Amplification (e.g., by PCR) of the resulting total EGFRcDNA (e.g., wild-type EGFR cDNA and EGFRvIII cDNA) is directed byreverse primer RPtot in combination with forward primer FPtot, which isspecific to EGFR exon 29. The fluorescently labeled probe (PBtot)targets EGFR exon 29 sequences between the forward and reverse primersand thus provides for the detection of total EGFR amplification product(e.g., by real-time PCR), e.g., from both wild-type EGFR RNA andEGFRvIII RNA.

TABLE 2 Total EGFR oligonucleotides SEQ ID name description target sitesequence (5′ to 3′) NO RPtot total EGFR reverse splice junction of EGFRGGGAACGGACTGGTTTATGTATTcag  7 primer exon 29 and EGFR exon 30 FPtottotal EGFR forward EGFR exon 29 CGCCTTGACTGAGGACAGCATAG  8 primer PBtottotal EGFR probe EGFR exon 29 D-ACGACACCTTCCTC-Q  9 PBtottotal EGFR probe EGFR exon 29 D-A7A C76 67C 67C 7AG 6G-Q 32

In TABLE 2, upper case letters in the reverse primer sequence RPtot(e.g., GGGAACGGACTGGTTTATGTATT (SEQ ID NO: 10)) indicate the bases ofthe primer that hybridize to the 5′ end of EGFR exon 30 and lower caseletters in the reverse primer sequence RPtot (e.g., cag (SEQ ID NO: 11))indicate the bases of the primer that hybridize to the 3′ end of EGFRexon 29. In the total EGFR probe provided by SEQ ID NO: 32, a “6”indicates a 5-propynyl dU base and a “7” indicates a 5-methyl dC base.

Further, the probe sequence provided in TABLE 2 is derived from thesense strand of the EGFR gene, but the technology also encompasses useof a probe having a sequence that is complementary to the sequence ofPBtot provided in TABLE 2 (e.g., GAGGAAGGTGTCGT (SEQ ID NO: 12)).Embodiments provide that the dye D in probe PBtot is any moiety (e.g., adye) that can fluoresce and allow effective association of the probe tothe target. Embodiments provide that the quencher Q in probe PBtot atthe opposite end from the dye D in probe PBtot is any moiety thateffectively quenches the fluorescent dye D and allows effectiveassociation (e.g., hybridization) of these probes to their targetsequences. In TABLE 2, the dye D is attached to PBtot at the 5′ end ofPBtot and the quencher Q is attached to PBtot at the 3′ end of PBtot;however, the technology also includes a probe in which the dye D isattached to the probe at the 3′ end of the probe and the quencher Q isattached to the probe at the 5′ end of the probe. The probe in TABLE 2does not limit the technology. For instance, the technology alsoprovides embodiments in which a probe comprises a detectable label atthe 5′ end and/or at the 3′ end, e.g., a probe that comprises a dye D atthe 5′ end or at the 3′ end. Some embodiments provide a probe that doesnot comprise a quencher. In some embodiments, the primers and/or theprobe are modified, e.g., to include a variety of binding enhancers suchas a minor groove binder (“MGB”) or a pdU or pdC nucleotide.

Beta-Actin Internal Control Gene Amplification Primers and HybridizationProbes

In some embodiments, the technology provided herein relates to theamplification and detection of RNA from an endogenous gene for use as aninternal control. For example, during the development of embodiments ofthe technology described herein, two oligonucleotide primers (“RPact”and “FPact”) and one hybridization probe (“PBact”) were developed thattarget a house-keeping gene, e.g., beta-actin. Exemplary primer andprobe sequences contemplated for use in the technology are provided inTABLE 3; see, e.g., FIG. 3 and FIG. 4. As used herein, the terms“FPact”, “RPact”, and “PBact” refer to forward primers, reverse primers,and hybridization probes targeting beta-actin (ACTB) RNA or beta-actin(ACTB) cDNA as described herein and as exemplified by the specificprimers and probes contemplated and described.

These primers and probe find use in the amplification (e.g., by RT-PCR)and detection of beta-actin RNA. In particular, the reverse primer RPactanneals to a specific sequence spanning the splice junction ofbeta-actin exon 1 and beta-actin exon 2 (see, e.g., FIG. 3 and FIG. 4).Since RPact spans an exon/exon splice junction, RPact annealsspecifically to beta-actin mRNA and thus provides an amplificationproduct (e.g., a cDNA) from beta-actin mRNA, but RPact does not annealto beta-actin genomic DNA and thus does not provide an amplificationproduct from beta-actin genomic DNA. Amplification (e.g., by PCR) of thebeta-actin cDNA is directed by reverse primer RPact in combination withthe beta-actin forward primer FPact, which is specific to a sequence inthe beta-actin exon 1. The beta-actin hybridization probe (PBact)targets beta-actin exon 1 sequences between FPact and RPact, therebyproviding for the detection of the beta-actin amplicon (e.g., byreal-time PCR) (see, e.g., FIG. 3 and FIG. 4).

TABLE 3 beta-actin oligonucleotides name description target sitesequence (5′ to 3′) SEQ ID NO RPact beta-actin reversesplice junction of beta- TCATCATCCATGGTGAGctggc 13 primeractin exon 1 and beta- actin exon 2 FPact beta-actin forwardbeta-actin exon 1 GAGCACAGAGCCTCGCCTTTG 14 primer PBact beta-actin probebeta-actin exon 1 D-ATCCGCCGCCCGTCCACACC- 15 Q

In TABLE 3, upper case letters in the reverse primer sequence RPact(e.g., TCATCATCCATGGTGAG (SEQ ID NO: 16)) indicate the bases of theprimer that hybridize to the 5′ end of beta-actin exon 2 and lower caseletters in the reverse primer sequence RPact (e.g., ctggc (SEQ ID NO:17)) indicate the bases of the primer that hybridize to the 3′ end ofbeta-actin exon 1. Further, the probe sequence provided in TABLE 3 isderived from the sense strand of the beta-actin gene, but the technologyalso encompasses use of a probe having a sequence that is complementaryto the sequence of PBact provided in TABLE 3 (e.g., GGTGTGGACGGGCGGCGGAT(SEQ ID NO: 18)). Embodiments provide that the dye D in probe PBact isany moiety (e.g., a dye) that can fluoresce and allow effectiveassociation of the probe to the target. Embodiments provide that thequencher Q in probe PBact at the opposite end from the dye D in probePBact is any moiety that effectively quenches the fluorescent dye D andallows effective association (e.g., hybridization) of these probes totheir target sequences. In TABLE 3, the dye D is attached to PBact atthe 5′ end of PBact and the quencher Q is attached to PBact at the 3′end of PBact; however, the technology also includes a probe in which thedye D is attached to the probe at the 3′ end of the probe and thequencher Q is attached to the probe at the 5′ end of the probe. Theprobe in TABLE 3 does not limit the technology. For instance, thetechnology also provides embodiments in which a probe comprises adetectable label at the 5′ end and/or at the 3′ end, e.g., a probe thatcomprises a dye D at the 5′ end or at the 3′ end. Some embodimentsprovide a probe that does not comprise a quencher. In some embodiments,the primers and/or the probe are modified, e.g., to include a variety ofbinding enhancers such as a minor groove binder (“MGB”) or a pdU or pdCnucleotide.

Alternative Internal Control Gene Amplification Primers andHybridization Probes

While the beta-actin primers and probe described herein are preferredfor certain embodiments of the technology, some embodiments of thetechnology provide other primer and probe sets for the amplification anddetection of RNA from two alternate endogenous control gene targets,e.g., abelson tyrosine kinase (ABL, also known as ABL1 and c-abl) andglucose-6-phoshate dehydrogenase (G6PD). During the development ofembodiments of the technology provided herein, two oligonucleotideprimers (“RPabl” and “FPabl”) and one hybridization probe (“PBabl”) weredeveloped that target the house-keeping gene ABL. Exemplary primer andprobe sequences contemplated for use in the technology are provided inTABLE 4. In addition, during the development of embodiments of thetechnology provided herein, two additional oligonucleotide primers(“RPg6pd” and “FPg6pd”) and one additional hybridization probe(“PBg6pd”) were developed that target the house-keeping gene G6PD.Exemplary primer and probe sequences contemplated for use in thetechnology are provided in TABLE 4. Accordingly, embodiments of thetechnology provide that the ACTB, ABL, and/or G6PD are used asendogenous control targets; accordingly, embodiments provide that theprimer/probe sets for ABL and/or G6PD are substituted for (orsupplement) the ACTB primer/probe set described hereinabove. As usedherein, the terms “FPabl”, “RPabl”, and “PBabl” refer to forwardprimers, reverse primers, and hybridization probes targeting ABL RNA orABL cDNA as described herein and as exemplified by the specific primersand probes contemplated and described. As used herein, the terms“FPg6pd”, “RPg6pd”, and “PBg6pd” refer to forward primers, reverseprimers, and hybridization probes targeting G6PD RNA or G6PD cDNA asdescribed herein and as exemplified by the specific primers and probescontemplated and described.

These primer and probe sets find use in the amplification (e.g., byRT-PCR) and detection of ABL RNA and/or G6PD RNA. In particular, reverseprimer RPabl anneals to a sequence within ABL exon 4 and directs thereverse transcription of ABL RNA (e.g., to produce ABL cDNA). Forwardprimer FPabl anneals to a sequence spanning the splice junction of ABLexon 3 and ABL exon 4. In the ABL gene, exon 3 is separated from exon 4by a large intron. Thus, use of forward primer FPabl in combination withreverse primer RPabl directs the amplification of ABL cDNA. The distancebetween the FPabl and RPabl binding sites in the genomic ABL genesequence prevents amplification from the ABL genomic DNA and thus FPabland RPabl do not provide an amplification product from ABL genomic DNA.Accordingly, this primer design provides amplicons from ABL RNA and/orABL cDNA, but does not produce amplicons from ABL genomic DNA. Thehybridization probe PBabl binds to ABL sequence between the ABL forwardand reverse primers FPabl and RPabl, and thereby provides for thedetection of the ABL amplicon.

Reverse primer RPg6pd anneals to a sequence within G6PD exon 3 anddirects reverse transcription of G6PD RNA. Forward primer FPg6pd annealsto a sequence within G6PD exon 2 and, in combination with reverse primerRPg6pd, provides for the amplification of the G6PD cDNA. The resultingamplicon spans sequences from G6PD exon 2 and G6PD exon 3. In the G6PDgene, exon 2 is separated from exon 3 by a large intron. Thus, use offorward primer FPg6pd in combination with reverse primer RPg6pd directsthe amplification of G6PD cDNA. The distance between the FPg6pd andRPg6pd binding sites in the genomic G6PD gene sequence preventsamplification from the G6PD genomic DNA and thus FPg6pd and RPg6pd donot provide an amplification product from G6PD genomic DNA. Accordingly,this primer design provides amplicons derived from G6PD RNA and/or G6PDcDNA, but does not produce amplicons from G6PD genomic DNA.Hybridization probe PBg6pd binds to a sequence at the junction of G6PDexon 2 and G6PD exon 3, and thereby provides for the detection of theG6PD amplicon. G6PD exon 2 is separated from G6PD exon 3 by an intron inthe genomic sequence; thus, the G6PD exon 2/G6PD exon 3 junctionsequence targeted by PBg6pd is specific to G6PD RNA and is not presentin the G6PD DNA. Accordingly, the G6PD signal detected by the PBg6pdprobe is derived from G6PD RNA and/or G6PD cDNA, and is not from G6PDgenomic DNA.

TABLE 4 ABL and G6PD oligonucleotides SEQ ID name descriptiontarget site sequence (5′ to 3′) NO RPabl ABL reverse primer ABL exon 4ACCGTTGAATGATGATGAACCAACT 19 FPabl ABL forward primer ABL exon 3AACACTGCTTCTGATGGCAAGct 20 PBabl ABL probe ABL exon 4D-CTCCTCCGAGAGCCGCTTCAACACC-Q 21 RPg6pd G6PD reverse primer G6PD exon 3AGATGGTGGGGTAGATCTTCTTCTTG 22 FPg6pd G6PD forward primer G6PD exon 2ATGCCTTCCATCAGTCGGATACA 23 PBg6pd G6PD probe splice junction ofD-CATGGGTGCATCGggtgacctg-Q 24 G6PD exon 2 and G6PD exon 3

In TABLE 4, upper case letters in the forward primer sequence FPabl(e.g., AACACTGCTTCTGATGGCAAG (SEQ ID NO: 25)) indicate the bases of theprimer that hybridize to the 3′ end of ABL exon 3 and lower case lettersin the reverse primer sequence RPabl (e.g., ct (SEQ ID NO: 26)) indicatethe bases of the primer that hybridize to the 5′ end of ABL exon 4.Upper case letters in the probe sequence PBg6pd (e.g., CATGGGTGCATCG(SEQ ID NO: 27)) indicate the bases of the probe that hybridize to the3′ end of G6PD exon 2 and lower case letters in the probe sequencePBg6pd (e.g., ggtgacctg (SEQ ID NO: 28)) indicate the bases of the probethat hybridize to the 5′ end of G6PD exon 3. Further, the probesequences for PBabl and PBg6pd provided in TABLE 4 are derived from thesense strands of the ABL gene and the G6PD gene, respectively, but thetechnology also encompasses use of a probe having a sequence that iscomplementary to the sequence of PBabl provided in TABLE 4 (e.g.,GGTGTTGAAGCGGCTCTCGGAGGAG (SEQ ID NO: 29)) and use of a probe having asequence that is complementary to the sequence of PBg6pd provided inTABLE 4 (e.g., caggtcaccCGATGCACCCATG (SEQ ID NO: 30)).

Embodiments provide that the dye D in probe PBabl and/or in probe PBg6pdis any moiety (e.g., a dye) that can fluoresce and allow effectiveassociation of the probe to the target. Embodiments provide that thequencher Q in probe PBabl and/or in probe PBg6pd at the opposite endfrom the dye D in probe PBabl and/or in probe PBg6pd is any moiety thateffectively quenches the fluorescent dye D and allows effectiveassociation (e.g., hybridization) of these probes to their targetsequences. In TABLE 4, the dye D is attached to PBabl and/or PBg6pd atthe 5′ end of PBabl and/or PBg6pd and the quencher Q is attached toPBabl and/or PBg6pd at the 3′ end of PBabl and/or PBg6pd; however, thetechnology also includes probes in which the dye D is attached to theprobe at the 3′ end of the probe and the quencher Q is attached to theprobe at the 5′ end of the probe. The probes in TABLE 4 do not limit thetechnology. For instance, the technology also provides embodiments inwhich a probe comprises a detectable label at the 5′ end and/or at the3′ end, e.g., a probe that comprises a dye D at the 5′ end or at the 3′end. Some embodiments provide a probe that does not comprise a quencher.In some embodiments, the primers and/or the probe are modified, e.g., toinclude a variety of binding enhancers such as a minor groove binder(“MGB”) or a pdU or pdC nucleotide.

A summary of the exemplary contemplated and described primers and probes(e.g., primer sequences and probe sequences) is provided in TABLE 5.

TABLE 5 Exemplary sequences of primers and probes oligo name short nameoligo type sequence (5′ to 3′) SEQ ID NO: EGvIIIi1_-58to-41 FPvIIIprimer CTCCTGGCGCTGCTGGCT  2 EGvIII-A primerCGTGATCTGTCACCACATAATTACCTTTC 33 EGvIIIi1_-39to-21pFAM PBvIII probeCGCTCTGCCCGGCGAGTCG  3 EGFRvIII Rev_D RPvIII primer ACCACATAATTACctttc 1 EFFRvIII Rev RPvIII primer CGTGATCTGTCACCACATAATTACctttc 31EGwti7_-28to-9 FPwtA primer GGTGCCACCTGCGTGAAGAA 34 EGv3i1_+36to+18RPvIIIB primer GGACGCACGAGCCGTGATC 35 EGFRwti7_-7to+8 NED MGB RPwtBprobe TGTCCCCGTAATTAT 36 EGwti29_-47to-25 FPtot primerCGCCTTGACTGAGGACAGCATAG  8 EGwti29_+23to-3 RPtot primerGGGAACGGACTGGTTTATGTATTcag  7 EGi29_-24to-11pNED PBtot probeACGACACCTTCCTC  9 Total EGFR Probe PBtot probe A7A C76 67C 67C 7AG 6G 32bActl1_-53to-33 FPact primer GAGCACAGAGCCTCGCCTTTG 14bActl1_-29to-10pCY5 PBact probe ATCCGCCGCCCGTCCACACC 15 bActl1_+17to-5RPact primer TCATCATCCATGGTGAGctggc 13 ABLi3_fwd-21to+2 FPabl primerAACACTGCTTCTGATGGCAAGct 20 ABLi3+65to+41 RPabl primerACCGTTGAATGATGATGAACCAACT 19 ABLi3+9to+33p PBabl probeCTCCTCCGAGAGCCGCTTCAACACC 21 G6PDi2_fwd-50to-28 primerATGCCTTCCATCAGTCGGATACA 37 G6PDi2fwd-57to-39 FPg6pd primerATGCCTTCCATCAGTCGGATACA 23 G6PDi2_rev-37to-12 RPg6pd primerAGATGGTGGGGTAGATCTTCTTCTTG 22 G6PDv1_fwd346to367pCY5 PBg6pd probeCATGGGTGCATCGggtgacctg 24

In TABLE 5, probes with names comprising “NED”, “FAM”, and “CY5”indicate an oligonucleotide that comprises in some embodiments a NED,FAM, and CY5 fluorescent moiety, respectively. Probes with namescomprising “MGB” indicate an oligonucleotide that comprises in someembodiments a minor groove binder. In the total EGFR probe provided bySEQ ID NO: 32, a “6” indicates a 5-propynyl dU base and a “7” indicatesa 5-methyl dC base.

Exemplary Compositions and Reaction Mixtures

Some embodiments provide a reaction mixture comprising a primer and/or aprobe for the detection of EGFRvIII expression and/or for detection ofEGFR expression. Particular embodiments provide a reaction mixturecomprising a primer and/or a probe for the detection of EGFRvIII mRNAand/or for detection of EGFR mRNA. For example, some embodiments providea reaction mixture comprising a primer and/or a probe as described inTABLE 1, TABLE 2, TABLE 3, TABLE 4, and/or TABLE 5. Some embodimentsprovide a reaction mixture comprising one or more of the primers RPvIIIand/or FPvIII and the probe PBvIII; and/or one or more of the primersRPtot and/or FPtot and the probe PBtot.

Some embodiments of reaction mixtures further comprise primers and/orprobes for the detection of an internal (e.g., endogenous) control(e.g., a gene such as ACTB, ABL, and/or G6PD). For example, someembodiments of reaction mixtures comprise one or more of the primersRPact and/or FPact and the probe PBact; one or more of the primers RPabland/or FPabl and the probe PBabl; and/or one or more of the primersRPg6pd and/or FPg6pd and the probe PBg6pd. In an exemplary embodiment, areaction mixture comprises RPvIII, FPvIII, PBvIII, RPact, FPact, andPBact. In an exemplary embodiment, a reaction mixture comprises RPtot,FPtot, PBtot, RPact, FPact, and PBact. In an exemplary embodiment, areaction mixture comprises RPvIII, FPvIII, PBvIII, RPtot, FPtot, PBtot,RPact, FPact, and PBact.

Embodiments of reaction mixtures comprise one or more of theabove-mentioned oligonucleotide primers and probes (e.g., a primerand/or a probe for the detection of EGFRvIII mRNA and/or EGFRvIIIexpression and/or for detection of EGFR mRNA and/or EGFR expression) andcomponents associated with nucleic acid amplification, such as dNTP mix(e.g., a mixture of dATP, dCTP, dGTP, and/or dTTP), buffer, polymeraseenzyme (e.g., a thermostable polymerase such as Taq, Tth, Pfu, etc.),and/or divalent ion (e.g., Mg²⁺ and/or Mn²⁺) as activation reagent.

Some embodiments provide a reaction mixture comprising primers. Forexample, some embodiments provide a reaction mixture comprising a primerfor the detection of EGFRvIII expression and/or for detection of EGFRexpression. Embodiments provide a reaction mixture comprising a primerfor the detection of EGFRvIII mRNA and/or for detection of EGFR mRNA.For example, some embodiments provide a reaction mixture comprising aprimer as described in TABLE 1, TABLE 2, TABLE 3, TABLE 4, and/or TABLE5. Alternatively, some embodiments comprise providing a reaction mixturecomprising one or more of the primers RPvIII and/or FPvIII; and/or oneor more of the primers RPtot and/or FPtot.

Some embodiments provide a reaction mixture comprising primers for thedetection of an internal (e.g., endogenous) control (e.g., a gene suchas ACTB, ABL, and/or G6PD). That is, some embodiments provide a reactionmixture that does not comprise a probe (e.g., some embodiments providethat a probe is added in later subsequent steps). For example, someembodiments provide a reaction mixture comprising one or more of theprimers RPact and/or FPact; one or more of the primers RPabl and/orFPabl; and/or one or more of the primers RPg6pd and/or FPg6pd. In anexemplary embodiment, a reaction mixture comprises RPvIII, FPvIII,RPact, and FPact. In an exemplary embodiment, a reaction mixturecomprises RPtot, FPtot, RPact, and FPact. In an exemplary embodiment, areaction mixture comprises RPvIII, FPvIII, RPtot, FPtot, RPact, andFPact.

Embodiments provide a reaction mixture comprising one or more of theabove-mentioned oligonucleotide primers (e.g., a primer for thedetection of EGFRvIII mRNA and/or EGFRvIII expression and/or fordetection of EGFR mRNA and/or EGFR expression) and components associatedwith nucleic acid amplification, such as dNTP mix (e.g., a mixture ofdATP, dCTP, dGTP, and/or dTTP), buffer, polymerase enzyme (e.g., athermostable polymerase such as Taq, Tth, Pfu, etc.), and/or divalention (e.g., Mg²⁺ and/or Mn²⁺) as activation reagent.

Further embodiments provide an assay mixture comprising a reactionmixture as described above and a test sample. In particular embodiments,the test sample is a sample in need of testing for one or more ofEGFRvIII mRNA presence or quantity, EGFRvIII expression level, EGFR mRNApresence or quantity, and/or EGFR expression level. In some embodiments,the test sample is RNA (e.g., total RNA) purified from a biologicalsample (e.g., cells, tissues, etc. from an organism). Accordingly, insome embodiments the sample (e.g., and the assay mixture) comprisespurified RNA (e.g., from a biological sample) and thus potentiallycomprises EGFRvIII transcripts (e.g., mRNA) and/or total EGFRtranscripts (e.g., mRNA). In some embodiments, an assay mixture istested to identify the sample as comprising or not comprising (e.g.,lacking) EGFRvIII mRNA. In some embodiments, a sample “not comprising”or “lacking” EGFPvIII mRNA is a sample in which EGFPvIII mRNA is absent,a sample in which EGFPvIII mRNA is undetectable, and/or a sample inwhich EGFPvIII mRNA amount is below a detection threshold.

As discussed above, some embodiments of the technology relate toreaction mixtures for detecting EGFRvIII expression (e.g., for detectingEGFRvIII mRNA or EGFRvIII cDNA) and/or for detecting and/or quantifyingtotal EGFR expression (e.g., for detecting and/or quantifying total EGFRmRNA or cDNA produced from total EGFR mRNA). Some embodiments of thetechnology relate to reaction mixtures for detecting EGFRvIII expression(e.g., for detecting EGFRvIII mRNA or EGFRvIII cDNA) and/or fordetecting and/or quantifying total EGFR expression (e.g., for detectingand/or quantifying total EGFR mRNA or cDNA produced from total EGFRmRNA) relative to the expression of a control mRNA such as, e.g., ACTB,ABL, or G6PD.

For instance, some embodiments provide a reaction mixture comprising aset of primers for detecting EGFRvIII mRNA, e.g., an oligonucleotidecomprising, consisting of, or consisting essentially of a sequenceaccording to SEQ ID NO: 1 or SEQ ID NO: 31 (e.g., an oligonucleotidecomprising, consisting of, or consisting essentially of a sequence or areverse complement of a sequence that is at least 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to SEQ ID NO: 1 or SEQ ID NO: 31 or that is 100%identical to SEQ ID NO: 1 or SEQ ID NO: 31) and an oligonucleotidecomprising, consisting of, or consisting essentially of a sequenceaccording to SEQ ID NO: 2 (e.g., an oligonucleotide comprising,consisting of, or consisting essentially of a sequence or a reversecomplement of a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 2 or that is 100% identical to SEQ ID NO: 2). Insome embodiments, reaction mixtures further comprise a probe fordetecting EGFRvIII, e.g., an oligonucleotide comprising, consisting of,or consisting essentially of a sequence according to SEQ ID NO: 3 (e.g.,an oligonucleotide comprising, consisting of, or consisting essentiallyof a sequence or a reverse complement of a sequence that is at least80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 3 or that is100% identical to SEQ ID NO: 3). In some embodiments, theoligonucleotide comprising, consisting of, or consisting essentially ofa sequence according to SEQ ID NO: 3 comprises a detectable label (e.g.,a fluorescent moiety). In some embodiments, the oligonucleotidecomprising, consisting of, or consisting essentially of a sequenceaccording to SEQ ID NO: 3 comprises a detectable label (e.g., afluorescent moiety) and a quencher.

Some embodiments of the technology relate to reaction mixtures fordetecting EGFRvIII expression (e.g., for detecting EGFRvIII mRNA orcDNA) and/or for detecting and/or quantifying total EGFR expression(e.g., for detecting and/or quantifying total EGFR mRNA or cDNA producedfrom total EGFR mRNA). For instance, some embodiments provide a reactionmixture comprising a set of primers for detecting EGFRvIII mRNA, e.g.,an oligonucleotide comprising, consisting of, or consisting essentiallyof a sequence according to SEQ ID NO: 1 or SEQ ID NO: 31 (e.g., anoligonucleotide comprising, consisting of, or consisting essentially ofa sequence or a reverse complement of a sequence that is at least 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1 or SEQ ID NO: 31 orthat is 100% identical to SEQ ID NO: 1 or SEQ ID NO: 31) and anoligonucleotide comprising, consisting of, or consisting essentially ofa sequence according to SEQ ID NO: 2 (e.g., an oligonucleotidecomprising, consisting of, or consisting essentially of a sequence or areverse complement of a sequence that is at least 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to SEQ ID NO: 2 or that is 100% identical to SEQID NO: 2). In some embodiments, reaction mixtures further comprise aprobe for detecting EGFRvIII, e.g., an oligonucleotide comprising,consisting of, or consisting essentially of a sequence according to SEQID NO: 3 (e.g., an oligonucleotide comprising, consisting of, orconsisting essentially of a sequence or a reverse complement of asequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical toSEQ ID NO: 3 or that is 100% identical to SEQ ID NO: 3). In someembodiments, the oligonucleotide comprising, consisting of, orconsisting essentially of a sequence according to SEQ ID NO: 3 comprisesa detectable label (e.g., a fluorescent moiety). In some embodiments,the oligonucleotide comprising, consisting of, or consisting essentiallyof a sequence according to SEQ ID NO: 3 comprises a detectable label(e.g., a fluorescent moiety) and a quencher. And, in some embodiments,reaction mixtures further comprise a set of primers for detecting and/orquantifying total EGFR mRNA, e.g., an oligonucleotide comprising,consisting of, or consisting essentially of a sequence according to SEQID NO: 7 (e.g., an oligonucleotide comprising, consisting of, orconsisting essentially of a sequence or a reverse complement of asequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical toSEQ ID NO: 7 or that is 100% identical to SEQ ID NO: 7) and anoligonucleotide comprising, consisting of, or consisting essentially ofa sequence according to SEQ ID NO: 8 (e.g., an oligonucleotidecomprising, consisting of, or consisting essentially of a sequence or areverse complement of a sequence that is at least 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to SEQ ID NO: 8 or that is 100% identical to SEQID NO: 8). In some embodiments, reaction mixtures further comprise aprobe for detecting and/or quantifying total EGFR, e.g., anoligonucleotide comprising, consisting of, or consisting essentially ofa sequence according to SEQ ID NO: 9 or SEQ ID NO: 32 or SEQ ID NO: 32(e.g., an oligonucleotide comprising, consisting of, or consistingessentially of a sequence or a reverse complement of a sequence that isat least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 9 orSEQ ID NO: 32 or SEQ ID NO: 32 or that is 100% identical to SEQ ID NO: 9or SEQ ID NO: 32 or SEQ ID NO: 32). In some embodiments, theoligonucleotide comprising, consisting of, or consisting essentially ofa sequence according to SEQ ID NO: 9 or SEQ ID NO: 32 or SEQ ID NO: 32comprises a detectable label (e.g., a fluorescent moiety). In someembodiments, the oligonucleotide comprising, consisting of, orconsisting essentially of a sequence according to SEQ ID NO: 9 or SEQ IDNO: 32 or SEQ ID NO: 32 comprises a detectable label (e.g., afluorescent moiety) and a quencher.

Some embodiments of the technology relate to reaction mixtures fordetecting and/or quantifying total EGFR expression. In some embodiments,reaction mixtures further comprise a set of primers for detecting and/orquantifying total EGFR mRNA, e.g., an oligonucleotide comprising,consisting of, or consisting essentially of a sequence according to SEQID NO: 7 (e.g., an oligonucleotide comprising, consisting of, orconsisting essentially of a sequence or a reverse complement of asequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical toSEQ ID NO: 7 or that is 100% identical to SEQ ID NO: 7) and anoligonucleotide comprising, consisting of, or consisting essentially ofa sequence according to SEQ ID NO: 8 (e.g., an oligonucleotidecomprising, consisting of, or consisting essentially of a sequence or areverse complement of a sequence that is at least 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to SEQ ID NO: 8 or that is 100% identical to SEQID NO: 8). In some embodiments, reaction mixtures further comprise aprobe for detecting and/or quantifying total EGFR, e.g., anoligonucleotide comprising, consisting of, or consisting essentially ofa sequence according to SEQ ID NO: 9 or SEQ ID NO: 32 or SEQ ID NO: 32(e.g., an oligonucleotide comprising, consisting of, or consistingessentially of a sequence or a reverse complement of a sequence that isat least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 9 orSEQ ID NO: 32 or that is 100% identical to SEQ ID NO: 9 or SEQ ID NO:32). In some embodiments, the oligonucleotide comprising, consisting of,or consisting essentially of a sequence according to SEQ ID NO: 9 or SEQID NO: 32 comprises a detectable label (e.g., a fluorescent moiety). Insome embodiments, the oligonucleotide comprising, consisting of, orconsisting essentially of a sequence according to SEQ ID NO: 9 or SEQ IDNO: 32 comprises a detectable label (e.g., a fluorescent moiety) and aquencher.

Further embodiments of reaction mixtures comprise a set of primers and aprobe for the detection and/or quantification of a control mRNA such asACTB, ABL, or G6PD. Thus, in some embodiments reaction mixtures compriseprimers and a probe to detect ACTB, e.g., an oligonucleotide comprising,consisting of, or consisting essentially of a sequence according to SEQID NO: 13 (e.g., an oligonucleotide comprising, consisting of, orconsisting essentially of a sequence or a reverse complement of asequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical toSEQ ID NO: 13 or that is 100% identical to SEQ ID NO: 13), anoligonucleotide comprising, consisting of, or consisting essentially ofa sequence according to SEQ ID NO: 14 (e.g., an oligonucleotidecomprising, consisting of, or consisting essentially of a sequence or areverse complement of a sequence that is at least 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to SEQ ID NO: 14 or that is 100% identical to SEQID NO: 14), and a probe comprising an oligonucleotide sequence accordingto SEQ ID NO: 15 (e.g., an oligonucleotide comprising, consisting of, orconsisting essentially of a sequence or a reverse complement of asequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical toSEQ ID NO: 15 or that is 100% identical to SEQ ID NO: 15). In someembodiments, reaction mixtures comprise primers and a probe to detectABL, e.g., an oligonucleotide comprising, consisting of, or consistingessentially of a sequence according to SEQ ID NO: 19 (e.g., anoligonucleotide comprising, consisting of, or consisting essentially ofa sequence or a reverse complement of a sequence that is at least 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 19 or that is 100%identical to SEQ ID NO: 19), an oligonucleotide comprising, consistingof, or consisting essentially of a sequence according to SEQ ID NO: 20(e.g., an oligonucleotide comprising, consisting of, or consistingessentially of a sequence or a reverse complement of a sequence that isat least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 20 orthat is 100% identical to SEQ ID NO: 20), and a probe comprising anoligonucleotide sequence according to SEQ ID NO: 21 (e.g., anoligonucleotide comprising, consisting of, or consisting essentially ofa sequence or a reverse complement of a sequence that is at least 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 21 or that is 100%identical to SEQ ID NO: 21). In some embodiments, reaction mixturescomprise primers and a probe to detect G6PD, e.g., an oligonucleotidecomprising, consisting of, or consisting essentially of a sequenceaccording to SEQ ID NO: 22 (e.g., an oligonucleotide comprising,consisting of, or consisting essentially of a sequence or a reversecomplement of a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 22 or that is 100% identical to SEQ ID NO: 22),an oligonucleotide comprising, consisting of, or consisting essentiallyof a sequence according to SEQ ID NO: 23 (e.g., an oligonucleotidecomprising, consisting of, or consisting essentially of a sequence or areverse complement of a sequence that is at least 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to SEQ ID NO: 23 or that is 100% identical to SEQID NO: 23), and a probe comprising an oligonucleotide sequence accordingto SEQ ID NO: 24 (e.g., an oligonucleotide comprising, consisting of, orconsisting essentially of a sequence or a reverse complement of asequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical toSEQ ID NO: 24 or that is 100% identical to SEQ ID NO: 24). In someembodiments, the oligonucleotide comprising, consisting of, orconsisting essentially of a sequence according to SEQ ID NO: 15, SEQ IDNO: 21, and/or SEQ ID NO: 24 comprises a detectable label (e.g., afluorescent moiety). In some embodiments, the oligonucleotidecomprising, consisting of, or consisting essentially of a sequenceaccording to SEQ ID NO: 15, SEQ ID NO: 21, and/or SEQ ID NO: 24comprises a detectable label (e.g., a fluorescent moiety) and aquencher.

Embodiments of reaction mixtures comprise one or more of theabove-mentioned oligonucleotide primers and/or probes (e.g., a primerand/or a probe for the detection of EGFRvIII mRNA and/or EGFRvIIIexpression and/or for detection of EGFR mRNA and/or EGFR expression) andcomponents associated with nucleic acid amplification, such as dNTP mix(e.g., a mixture of dATP, dCTP, dGTP, and/or dTTP), buffer, polymeraseenzyme (e.g., a thermostable polymerase such as Taq, Tth, Pfu, etc.),and/or divalent ion (e.g., Mg²⁺ and/or Mn²⁺) as activation reagent. Someembodiments provide reaction mixtures comprising only primers and someembodiments provide reaction mixtures comprising primers and probes.

Further embodiments provide an assay mixture comprising a reactionmixture as described above and a test sample. In particular embodiments,the test sample is a sample in need of testing for one or more ofEGFRvIII mRNA presence or quantity, EGFRvIII expression level, EGFR mRNApresence or quantity, and/or EGFR expression level. In some embodiments,the test sample is RNA (e.g., total RNA) purified from a biologicalsample (e.g., cells, tissues, etc. from an organism). Accordingly, insome embodiments the sample (e.g., and the assay mixture) comprisespurified RNA (e.g., from a biological sample) and thus potentiallycomprises EGFRvIII transcripts (e.g., mRNA) and/or total EGFRtranscripts (e.g., mRNA). In some embodiments, an assay mixture istested to identify the sample as comprising or not comprising (e.g.,lacking) EGFRvIII mRNA. In some embodiments, a sample “not comprising”or “lacking” EGFPvIII mRNA is a sample in which EGFPvIII mRNA is absent,a sample in which EGFPvIII mRNA is undetectable, and/or a sample inwhich EGFPvIII mRNA amount is below a detection threshold.

Exemplary Methods

In some embodiments, the technology provides a diagnostic method.Particular embodiments provide a method comprising steps for, e.g.,providing a reaction mixture comprising a primer and/or a probe for thedetection of EGFRvIII expression and/or for detection of EGFRexpression. Particular embodiments provide a reaction mixture comprisinga primer and/or a probe for the detection of EGFRvIII mRNA and/or fordetection of EGFR mRNA. For example, some embodiments provide a reactionmixture comprising a primer and/or a probe as described in TABLE 1,TABLE 2, TABLE 3, TABLE 4, and/or TABLE 5. Alternatively, someembodiments comprise providing a reaction mixture comprising one or moreof the primers RPvIII and/or FPvIII and the probe PBvIII; and/or one ormore of the primers RPtot and/or FPtot and the probe PBtot.

Some embodiments of methods further comprise providing a reactionmixture comprising primers and/or probes for the detection of aninternal (e.g., endogenous) control (e.g., a gene such as ACTB, ABL,and/or G6PD). For example, some embodiments of methods compriseproviding a reaction mixture comprising one or more of the primers RPactand/or FPact and the probe PBact; one or more of the primers RPabland/or FPabl and the probe PBabl; and/or one or more of the primersRPg6pd and/or FPg6pd and the probe PBg6pd. In an exemplary embodiment,methods comprise a step of providing a reaction mixture comprisingRPvIII, FPvIII, PBvIII, RPact, FPact, and PBact. In an exemplaryembodiment, methods comprise a step of providing a reaction mixturecomprising RPtot, FPtot, PBtot, RPact, FPact, and PBact. In an exemplaryembodiment, methods comprise a step of providing a reaction mixturecomprising RPvIII, FPvIII, PBvIII, RPtot, FPtot, PBtot, RPact, FPact,and PBact.

Embodiments of methods comprise providing a reaction mixture comprisingone or more of the above-mentioned oligonucleotide primers and probes(e.g., a primer and/or a probe for the detection of EGFRvIII mRNA and/orEGFRvIII expression and/or for detection of EGFR mRNA and/or EGFRexpression) and components associated with nucleic acid amplification,such as dNTP mix (e.g., a mixture of dATP, dCTP, dGTP, and/or dTTP),buffer, polymerase enzyme, and/or divalent ion as activation reagent.

Some embodiments provide a reaction mixture comprising primers. Forexample, embodiments provide a method comprising steps for, e.g.,providing a reaction mixture comprising a primer for the detection ofEGFRvIII expression and/or for detection of EGFR expression. Embodimentsprovide a reaction mixture comprising a primer for the detection ofEGFRvIII mRNA and/or for detection of EGFR mRNA. For example, someembodiments provide a reaction mixture comprising a primer as providedin TABLE 1, TABLE 2, TABLE 3, TABLE 4, and/or TABLE 5. Alternatively,some embodiments comprise providing a reaction mixture comprising one ormore of the primers RPvIII and/or FPvIII; and/or one or more of theprimers RPtot and/or FPtot.

Some embodiments of methods further comprise providing a reactionmixture comprising primers for the detection of an internal (e.g.,endogenous) control (e.g., a gene such as ACTB, ABL, and/or G6PD). Forexample, some embodiments of methods comprise providing a reactionmixture comprising one or more of the primers RPact and/or FPact; one ormore of the primers RPabl and/or FPabl; and/or one or more of theprimers RPg6pd and/or FPg6pd. In an exemplary embodiment, methodscomprise a step of providing a reaction mixture comprising RPvIII,FPvIII, RPact, and FPact. In an exemplary embodiment, methods comprise astep of providing a reaction mixture comprising RPtot, FPtot, RPact, andFPact. In an exemplary embodiment, methods comprise a step of providinga reaction mixture comprising RPvIII, FPvIII, RPtot, FPtot, RPact, andFPact.

Embodiments of methods comprise providing a reaction mixture comprisingone or more of the above-mentioned oligonucleotide primers (e.g., aprimer for the detection of EGFRvIII mRNA and/or EGFRvIII expressionand/or for detection of EGFR mRNA and/or EGFR expression) and componentsassociated with nucleic acid amplification, such as dNTP mix (e.g., amixture of dATP, dCTP, dGTP, and/or dTTP), buffer, polymerase enzyme,and/or divalent ion as activation reagent.

Further embodiments provide a step of adding a test sample to anembodiment of a reaction mixture as described above to provide an assaymixture. In particular embodiments, the test sample is a sample in needof testing for one or more of EGFRvIII mRNA presence or quantity,EGFRvIII expression level, EGFR mRNA presence or quantity, and/or EGFRexpression level. In some embodiments, the test sample is RNA (e.g.,total RNA) purified from a biological sample (e.g., cells, tissues, etc.from an organism). Accordingly, in some embodiments the sample (e.g.,and the assay mixture) comprises purified RNA (e.g., from a biologicalsample) and thus potentially comprises EGFRvIII transcripts (e.g., mRNA)and/or total EGFR transcripts (e.g., mRNA). In some embodiments, it isan object of the methods provided to identify the sample as comprisingor not comprising (e.g., lacking) EGFRvIII mRNA. In some embodiments, asample “not comprising” or “lacking” EGFPvIII mRNA is a sample in whichEGFPvIII mRNA is absent, a sample in which EGFPvIII mRNA isundetectable, and/or a sample in which EGFPvIII mRNA amount is below adetection threshold.

In some embodiments, it is an object of the methods provided to quantifyEGFRvIII transcripts (e.g., mRNA) and/or to quantify total EGFRtranscripts (e.g., mRNA). In some embodiments, quantification provides aresult (e.g., a quantity or an amount) that is zero, greater than zero,or undetectable.

In some embodiments, methods comprise a reverse transcribing step, e.g.,a step of exposing the assay mixture to conditions appropriate forreverse transcription of the EGFRvIII RNA, total EGFR RNA, and/orendogenous control gene RNA using primers (e.g., forward and/or reverseprimers) of the assay mixture (e.g., as described above) to provide anRT-PCR product. In some embodiments, the RT-PCR product furthercomprises one or more cDNAs that are products of the reversetranscription of the EGFRvIII RNA, total EGFR RNA, and/or endogenouscontrol gene RNA. Accordingly, in some embodiments the assay mixturefurther comprises one or more cDNAs. In some embodiments, the RT-PCRproduct is used to provide an amplification reaction mixture (e.g., insome embodiments, an amplification reaction mixture is or comprises theRT-PCR product).

In some embodiments, methods comprise amplifying an amplificationreaction mixture (e.g., a mixture comprising the RT-PCR product (e.g., amixture comprising the one or more cDNAs of the RT-PCR product)) usingthe forward primers and reverse primers as described above to provide anamplicon. In some embodiments, the methods further comprise quantifyingand/or detecting the presence or absence of an amplicon with probes asdescribed above, e.g., quantifying and/or detecting the presence orabsence of amplified EGFRvIII, amplified total EGFR, and/or amplifiedendogenous control gene target sequences with probes as described above.

In some embodiments, the probes are provided in the assay mixture priorto reverse transcription and/or prior to amplification and, in someembodiments, probes are added to the amplicon after amplification.

In some embodiments, methods comprise a detecting step, e.g., adetecting step comprising measuring, assaying, determining, calculating,and/or comparing one or more of EGFRvIII expression level, EGFRvIIItranscript amount, total EGFR expression level, total EGFR transcriptamount, endogenous control gene (e.g., ACTB, ABL, G6PD) transcriptamount, and/or endogenous control gene (e.g., ACTB, ABL, G6PD)expression level.

Accordingly, in some embodiments the methods comprise measuring EGFRvIIIexpression, EGFRvIII transcript amount, total EGFR expression, and/ortotal EGFR transcript amount. In some embodiments measuring EGFRvIIIexpression and/or measuring EGFRvIII transcript amount provides aquantitative EGFRvIII result. In some embodiments measuring total EGFRexpression and/or total EGFR transcript amount provides a quantitativeEGFR result.

In some embodiments, a quantitative EGFRvIII result is a quantitativemeasure of EGFRvIII expression and/or a quantitative measure of EGFRvIIItranscript amount. In some embodiments, a quantitative EGFR resultprovides a quantitative measure of total EGFR expression and/or aquantitative measure of total EGFR transcript amount.

In some embodiments, the methods provide a qualitative result that ispresence or absence of EGFRvIII expression and/or a qualitative resultthat is presence or absence of EGFRvIII expression. In some embodiments,the methods comprise measuring an endogenous control gene (e.g., ACTB,ABL, G6PD) transcript level and/or measuring an endogenous control gene(e.g., ACTB, ABL, G6PD) expression level to provide a quantitativemeasure of an endogenous control gene (e.g., ACTB, ABL, G6PD) transcriptlevel and/or to provide a quantitative measure of endogenous controlgene (e.g., beta-actin, ABL, G6PD) expression level.

The technology provides embodiments of methods for determining EGFRvIIIexpression level, EGFRvIII transcript (e.g., mRNA) level, total EGFRexpression level, and/or total EGFR transcript (mRNA) level relative toendogenous control gene (e.g., ACTB, ABL, G6PD) transcript level and/orendogenous control gene (e.g., ACTB, ABL, G6PD) expression level, e.g.,to provide a relative EGFRvIII expression, a relative EGFRvIIItranscript amount, a relative total EGFR expression, and/or a relativetotal EGFR transcript amount. Accordingly, embodiments of the methodscomprise comparing a quantitative EGFRvIII or EGFR result (e.g., aquantitative measure of EGFRvIII expression, a quantitative measure ofEGFRvIII transcript amount, a quantitative measure of total EGFRexpression, and/or a quantitative measure of total EGFR transcriptamount) to a quantitative measure of an endogenous control gene (e.g.,ACTB, ABL, G6PD) transcript level and/or a quantitative measure of anendogenous control gene (e.g., ACTB, ABL, G6PD) expression level. Someembodiments comprise computing a ratio of a quantitative EGFRvIII orEGFR result (e.g., a quantitative measure of EGFRvIII expression, aquantitative measure of EGFRvIII transcript amount, a quantitativemeasure of total EGFR expression, and/or a quantitative measure of totalEGFR transcript amount) to a quantitative measure of an endogenouscontrol gene (e.g., ACTB, ABL, G6PD) transcript level and/or aquantitative measure of endogenous control gene (e.g., ACTB, ABL, G6PD)expression level. In some embodiments, the ratio is compared to a knownvalue (e.g., a value from a normal control) to provide an indication ordiagnosis, e.g., an indication or diagnosis of a cancer (e.g., anindication or diagnosis that the subject from whom the sample wasprocured has a cancer or has a risk of having a cancer).

In some embodiments, methods employ a polymerase enzyme or an enzyme mix(e.g., in a reaction mixture and/or in an assay mixture comprising areaction mixture and a sample) to catalyze both the reversetranscription of RNA sequences (e.g., by RT-PCR) and the amplificationof DNA (e.g., by amplification of cDNA produced in the RT-PCR product bythe reverse transcription of RNA in the assay mixture).

Accordingly, in some embodiments, the reverse transcribing step (e.g.,producing cDNA from RNA in the assay mixture) and the amplifying step(e.g., producing DNA amplicons from the cDNA in the RT-PCR product) areperformed in one tube (e.g., either simultaneously or sequentially),e.g., in a single-run, closed-tube format by an instrument capable ofconcurrent thermal cycling and signal (e.g., fluorescence) detection.Accordingly, in some embodiments the technology finds use in a real-timeamplification. In some embodiments, the technology comprises use of areal-time amplification apparatus.

In some embodiments, methods comprise use of multiple primer and/orprobe sets within a single assay mixture and/or within a singleamplification reaction mixture, e.g., during the reverse transcribingstep, during the amplification step, and/or during the detecting step.Accordingly, embodiments provide for the detection and quantification ofEGFRvIII RNA, total EGFR RNA, and the endogenous internal control genein a single reaction. For example, in some embodiments of the methodsprovided, the EGFRvIII primers and probe, the total EGFR primers andprobe, and the endogenous control primer and probe are used in multiplexwithin the same reaction. In some embodiments, each probe is labeledwith a different fluorescent dye to provide for differentiating theEGFRvIII, total EGFR, and endogenous control signals from each other(e.g., by detecting a different appropriate wavelength associated withthe emission spectrum of each fluorescent dye). In some embodiments,EGFRvIII mRNA and total EGFR mRNA levels in the sample are quantifiedrelative to the endogenous control RNA levels. Further, detection of theendogenous control RNA serves as a within-well sample validity control,e.g., as a control for cell adequacy, sample extraction, and/oramplification efficiency.

In some embodiments, the EGFRvIII primer/probe sets and the total EGFRprimer/probe sets function independently from each other. Accordingly,if relative quantification of only EGFRvIII expression (e.g., EGFRvIIImRNA levels) is of interest, the method is configured to provide areaction mixture comprising the EGFRvIII primer/probe set (e.g., byremoving the total EGFR primer/probe set). Alternatively, if relativequantification of only total EGFR expression (e.g., total EGFR mRNAlevels) is of interest, the method is configured to provide a reactionmixture comprising the total EGFR primer/probe set (e.g., by removingthe EGFRvIII primer/probe set).

In alternate embodiments of the methods provided, the EGFRvIIIprimer/probe, total EGFR primer/probe, and endogenous controlprimer/probe are used in separate reactions (e.g., in singleplex), eachreaction comprising an aliquot of the same RNA sample. Thus, in someembodiments the methods provide separate reaction mixtures comprisingthe EGFRvIII primer/probe, total EGFR primer/probe, and endogenouscontrol primer/probe (e.g., a first reaction mixture comprising theEGFRvIII primer/probe, a second reaction mixture comprising total EGFRprimer/probe, and a third reaction mixture comprising endogenous controlprimer/probe). Addition of an aliquot of a test sample (e.g., a samplein need of testing for one or more of EGFRvIII mRNA presence orquantity, EGFRvIII expression level, EGFR mRNA presence or quantity,and/or EGFR expression level (e.g., a test sample that comprises RNA(e.g., total RNA) purified from a biological sample (e.g., cells,tissues, etc. from an organism))) to each reaction mixture thus providesseparate assay mixtures (e.g., a first assay mixture comprising theEGFRvIII primer/probe, a second assay mixture comprising total EGFRprimer/probe, and a third assay mixture comprising endogenous controlprimer/probe) that are provided for reverse transcribing, amplifying,and detecting steps of the methods. In these embodiments, the EGFRvIIIRNA level and/or the total EGFR RNA level in the sample are quantifiedrelative to endogenous control RNA levels using between-well resultsfrom the independent EGFRvIII, total EGFR, and endogenous controlreactions.

In some embodiments, the EGFRvIII primer/probe set and the total EGFRprimer/probe set are used independently for qualitative (e.g.,presence/absence) detection of EGFRvIII mRNA and/or total EGFR mRNA in asample. In some embodiments, qualitative detection provides a resultindicating that EGFRvIII mRNA and/or total EGFR mRNA is/are greater thanor less than a threshold value. In some embodiments, the endogenouscontrol primer/probe set is used in combination with other primer/probesets (e.g., for detecting, quantifying, etc. EGFR or non-EGFR targets)to provide an endogenous internal control for relative quantification ofRNA levels from targeted genes and/or to provide a sample validitycontrol for cell adequacy, sample extraction, and/or amplificationefficiency.

Fluorescent Moieties and Quenchers

In some embodiments, an oligonucleotide (e.g., a probe) comprises adetectable label. In some embodiments, the detectable label is afluorescent moiety (e.g., a fluorogenic dye, also referred to as a“fluorophore” or a “fluor”). A wide variety of fluorescent moieties isknown in the art and methods are known for linking a fluorescent moietyto a nucleotide prior to incorporation of the nucleotide into anoligonucleotide and for adding a fluorescent moiety to anoligonucleotide after synthesis of the oligonucleotide.

Examples of compounds that may be used as the fluorescent moiety includebut are not limited to xanthene, anthracene, cyanine, porphyrin, andcoumarin dyes. Examples of xanthene dyes that find use with the presenttechnology include but are not limited to fluorescein,6-carboxyfluorescein (6-FAM), 5-carboxyfluorescein (5-FAM), 5- or6-carboxy-4, 7, 2′, 7′-tetrachlorofluorescein (TET), 5- or6-carboxy-4′5′2′4′5′7′ hexachlorofluorescein (HEX), 5′ or6′-carboxy-4′,5′-dichloro-2,′7′-dimethoxyfluorescein (JOE),5-carboxy-2′,4′,5′,7′-tetrachlorofluorescein (ZOE), rhodol, rhodamine,tetramethylrhodamine (TAMRA), 4,7-dlchlorotetramethyl rhodamine(DTAMRA), rhodamine X (ROX), VIC dye, NED dye, MAX dye, ATTO dyes, andTexas Red. Examples of cyanine dyes that find use in embodiments of thetechnology include but are not limited to Cy 3, Cy 3.5, Cy 5, Cy 5.5, Cy7, and Cy 7.5. Other fluorescent moieties and/or dyes that find use withthe present technology include but are not limited to energy transferdyes, composite dyes, and other aromatic compounds that give fluorescentsignals. In some embodiments, the fluorescent moiety comprises a quantumdot.

Fluorescent dyes include, without limitation, d-Rhodamine acceptor dyesincluding Cy5, dichloro[R110], dichloro[R6G], dichloro[TAMRA],dichloro[ROX] or the like; fluorescein donor dyes including fluorescein,6-FAM, 5-FAM, or the like; Acridine including Acridine orange, Acridineyellow, Proflavin, pH 7, or the like; Aromatic Hydrocarbons including2-Methylbenzoxazole, Ethyl p-dimethylaminobenzoate, Phenol, Pyrrole,benzene, toluene, or the like; Arylmethine Dyes including Auramine O,Crystal violet, glycerol, Malachite Green, or the like; Coumarin dyesincluding 7-Methoxycoumarin-4-acetic acid, Coumarin 1, Coumarin 30,Coumarin 314, Coumarin 343, Coumarin 6, or the like; Cyanine Dyesincluding 1,1′-diethyl-2,2′-cyanine iodide, Cryptocyanine,Indocarbocyanine (C3) dye, Indodicarbocyanine (C5) dye,Indotricarbocyanine (C7) dye, Oxacarbocyanine (C3) dye,Oxadicarbocyanine (C5) dye, Oxatricarbocyanine (C7) dye, Pinacyanoliodide, Stains all, Thiacarbocyanine (C3) dye, Thiacarbocyanine (C3)dye, Thiadicarbocyanine (C5) dye, Thiatricarbocyanine (C7) dye, or thelike; Dipyrrin dyes includingN,N′-Difluoroboryl-1,9-dimethyl-5-(4-iodophenyl)-dipyrrin,N,N′-Difluoroboryl-1,9-dimethyl-5-1(4-(2-trimethylsilylethynyl),N,N′-Difluoroboryl-1,9-dimethyl-5-phenydipyrrin, or the like;Merocyanines including4-(dicyanomethylene)-2-methyl-6-(p-dimethylaminostyryl)-4H-pyran (DCM),acetonitrile,4-(dicyanomethylene)-2-methyl-6-(p-dimethylaminostyryl)-4H-pyran (DCM),4-Dimethylamino-4′-nitrostilbene, Merocyanine 540, or the like;Miscellaneous Dyes including 4′,6-Diamidino-2-phenylindole (DAPI),dimethylsulfoxide, 7-Benzylamino-4-nitrobenz-2-oxa-1,3-diazole, Dansylglycine, Dansyl glycine, dioxane, Hoechst 33258, DMF, Hoechst 33258,Lucifer yellow CH, Piroxicam, Quinine sulfate, Quinine sulfate,Squarylium dye III, or the like; Oligophenylenes including2,5-Diphenyloxazole (PPO), Biphenyl, POPOP, p-Quaterphenyl, p-Terphenyl,or the like; Oxazines including Cresyl violet perchlorate, Nile Blue,Nile Red, Oxazine 1, Oxazine 170, or the like; Polycyclic AromaticHydrocarbons including 9,10-Bis(phenylethynyl) anthracene,9,10-Diphenylanthracene, Anthracene, Naphthalene, Perylene, Pyrene, orthe like; polyene/polyynes including 1,2-diphenylacetylene,1,4-diphenylbutadiene, 1,4-diphenylbutadiyne, 1,6-Diphenylhexatriene,Beta-carotene, Stilbene, or the like; Redox-active Chromophoresincluding Anthraquinone, Azobenzene, Benzoquinone, Ferrocene,Riboflavin, Tris(2,2′-bipyridypruthenium(II), Tetrapyrrole, Bilirubin,Chlorophyll a, diethyl ether, Chlorophyll a, Chlorophyll b,Diprotonated-tetraphenylporphyrin, Hematin, Magnesiumoctaethylporphyrin, Magnesium octaethylporphyrin (MgOEP), Magnesiumphthalocyanine (MgPc), PrOH, Magnesium phthalocyanine (MgPc), pyridine,Magnesium tetramesitylporphyrin (MgTMP), Magnesium tetraphenylporphyrin(MgTPP), Octaethylporphyrin, Phthalocyanine (Pc), Porphin, ROX, TAMRA,Tetra-t-butylazaporphine, Tetra-t-butylnaphthalocyanine,Tetrakis(2,6-dichlorophenyflporphyrin, Tetrakis(o-aminophenyflporphyrin,Tetramesitylporphyrin (TMP), Tetraphenylporphyrin (TPP), Vitamin B12,Zinc octaethylporphyrin (ZnOEP), Zinc phthalocyanine (ZnPc), pyridine,Zinc tetramesitylporphyrin (ZnTMP), Zinc tetramesitylporphyrin radicalcation, Zinc tetraphenylporphyrin (ZnTPP), or the like; Xanthenesincluding Eosin Y, Fluorescein, basic ethanol, Rhodamine 123, Rhodamine6G, Rhodamine B, Rose bengal, Sulforhodamine 101, or the like; ormixtures or combination thereof or synthetic derivatives thereof.

Several classes of fluorogenic dyes and specific compounds are knownthat are appropriate for particular embodiments of the technology:xanthene derivatives such as fluorescein, rhodamine, Oregon green,eosin, and Texas red; cyanine derivatives such as cyanine,indocarbocyanine, oxacarbocyanine, thiacarbocyanine, and merocyanine;naphthalene derivatives (dansyl and prodan derivatives); coumarinderivatives; oxadiazole derivatives such as pyridyloxazole,nitrobenzoxadiazole, and benzoxadiazole; pyrene derivatives such ascascade blue; oxazine derivatives such as Nile red, Nile blue, cresylviolet, and oxazine 170; acridine derivatives such as proflavin,acridine orange, and acridine yellow; arylmethine derivatives such asauramine, crystal violet, and malachite green; and tetrapyrrolederivatives such as porphin, phtalocyanine, bilirubin. In someembodiments the fluorescent moiety a dye that is xanthene, fluorescein,rhodamine, BODIPY, cyanine, coumarin, pyrene, phthalocyanine,phycobiliprotein, ALEXA FLUOR® 350, ALEXA FLUOR® 405, ALEXA FLUOR® 430,ALEXA FLUOR® 488, ALEXA FLUOR® 514, ALEXA FLUOR® 532, ALEXA FLUOR® 546,ALEXA FLUOR® 555, ALEXA FLUOR® 568, ALEXA FLUOR® 568, ALEXA FLUOR® 594,ALEXA FLUOR® 610, ALEXA FLUOR® 633, ALEXA FLUOR® 647, ALEXA FLUOR® 660,ALEXA FLUOR® 680, ALEXA FLUOR® 700, ALEXA FLUOR® 750, or a squarainedye. In some embodiments, the label is a fluorescently detectable moietyas described in, e.g., Haugland (September 2005) MOLECULAR PROBESHANDBOOK OF FLUORESCENT PROBES AND RESEARCH CHEMICALS (10th ed.), whichis herein incorporated by reference in its entirety.

In some embodiments the label (e.g., a fluorescently detectable label)is one available from ATTO-TEC GmbH (Am Eichenhang 50, 57076 Siegen,Germany), e.g., as described in U.S. Pat. Appl. Pub. Nos. 20110223677,20110190486, 20110172420, 20060179585, and 20030003486; and in U.S. Pat.No. 7,935,822, each of which is incorporated herein by reference.

One of ordinary skill in the art will recognize that dyes havingemission maxima outside these ranges may be used as well. In some cases,dyes ranging between 500 nm to 700 nm have the advantage of being in thevisible spectrum and can be detected using existing photomultipliertubes. In some embodiments, the broad range of available dyes allowsselection of dye sets that have emission wavelengths that are spreadacross the detection range. Detection systems capable of distinguishingmany dyes are known in the art.

In some embodiments, the technology comprises use of fluorescent dyesand/or molecules that quench (e.g., decrease, eliminate, and/orminimize) the fluorescence of another fluorescent dye. In someembodiments, an oligonucleotide comprises a quencher moiety. A widevariety of quencher moieties is known in the art. For example, in someembodiments an oligonucleotide comprises a quencher than is a Black HoleQuencher (e.g., BHQ-0, BHQ-1, BHQ-2, BHQ-3), a Dabcyl, an Iowa BlackQuencher (e.g., Iowa Black FQ, Iowa Black RQ), an Eclipse quencher.

In some embodiments a BHQ-1 is used with a fluorescent moiety that hasan emission wavelength from approximately 500-600 nm. In someembodiments a BHQ-2 is used with a fluorescent moiety that has anemission wavelength from approximately 550-675 nm. In some embodiments,a FRET pair is a fluorophore-quencher pair that provides quenching.

Some exemplary fluorophore-quencher pairs include FAM and BHQ-1, TET andBHQ-1, JOE and BHQ-1, HEX and BHQ-1, Cy3 and BHQ-2, TAMRA and BHQ-2, ROXand BHQ-2, Cy5 and BHQ-3, Cy5.5 and BHQ-3, FAM and BHQ-1, TET and BHQ-1,JOE and 3′-BHQ-1, HEX and BHQ-1, Cy3 and BHQ-2, TAMRA and BHQ-2, ROX andBHQ-2, Cy5 and BHQ-3, Cy5.5 and BHQ-3, or similar fluorophore-quencherpairs available from the commercial entities such as BiosearchTechnologies, Inc. of Novato, Calif.

Subjects

In some embodiments, the technology is related to testing a subject(e.g., a human). In some embodiments, a sample is obtained from thesubject for testing according to the technology provided. In someembodiments, the subject is in need of testing for the presence of acancer or a neoplasm. In some embodiments, the subject is in need oftesting to determine a risk of developing a cancer or a neoplasm.

In some embodiments, a subject is in need of testing to determine thelikelihood of responding to a therapy and/or medical intervention. Forexample, in some embodiments a subject is in need of testing todetermine the likelihood of responding to a therapy targeting EGFR(e.g., an anti-EGFR therapeutic agent, e.g., as described herein). Forexample, in some embodiments the subject has a tumor and the subject(e.g., the tumor) is tested to assess EGFRvIII presence and/or EGFRexpression to determine if the subject and/or the tumor is/are likely torespond to a therapy targeting EGFR (e.g., an anti-EGFR therapeuticagent). For example, in some embodiments, presence of EGFRvIII and/orEGFR overexpression indicates that the subject is likely to respond toan anti-EGFR therapy, e.g., an anti-EGFR therapy as described herein.

In some embodiments, the technology is related to diagnosing a subject(e.g., a human). In some embodiments, a sample is obtained from thesubject for testing according to the technology provided, e.g., toprovide information to diagnose the subject. In some embodiments, thesubject is in need of a diagnosis describing the presence or absence ofa cancer or a neoplasm. In some embodiments, the subject is in need of adiagnosis to determine a risk of developing a cancer or a neoplasm.

In some embodiments, the subject is diagnosed for the presence orabsence of a cancer that is lung cancer (e.g., non-small cell lungcancer), breast cancer, head and neck cancer, salivary gland cancer,colorectal cancer, pancreatic cancer, hepatocellular carcinoma,esophageal cancer, and/or glioblastoma.

In some embodiments, the subject is diagnosed for the risk of developinga cancer that is lung cancer (e.g., non-small cell lung cancer), breastcancer, head and neck cancer, salivary gland cancer, colorectal cancer,pancreatic cancer, hepatocellular carcinoma esophageal cancer, and/orglioblastoma.

In some embodiments, the subject does not have a mutation in KRAS. Insome embodiments, the subject has been treated with platinum-based orchemotherapy (e.g., docetaxel).

In some embodiments, a level of EGFR expression that is higher than thenormal level of EGFR expression (e.g., from a subject that does not havea cancer) indicates an increased EGFR expression. In some embodiments, alevel of EGFR expression that is 1.5×, 2×, 2.5×, 3×, 3.5×, 4×, 4.5×, 5×,6×, 7×, 8×, 9×, 10× or more than the normal level of EGFR expression(e.g., from a subject that does not have a cancer) indicates anincreased EGFR expression. In some embodiments, a level of EGFRexpression that is higher than the normal level of EGFR expression(e.g., from a subject that does not have a cancer) indicates that thesubject is in need of a treatment targeting EGFR (e.g., an anti-EGFRtherapeutic agent). In some embodiments, a level of EGFR expression thatis 1.5×, 2×, 2.5×, 3×, 3.5×, 4×, 4.5×, 5×, 6×, 7×, 8×, 9×, 10× or morethan the normal level of EGFR expression (e.g., from a subject that doesnot have a cancer) indicates that the subject is in need of a treatmenttargeting EGFR (e.g., an anti-EGFR therapeutic agent).

In some embodiments, detecting expression of EGFRvIII indicates that thesubject is in need of a treatment targeting EGFR (e.g., an anti-EGFRtherapeutic agent). In some embodiments, detecting expression ofEGFRvIII (e.g., detecting expression greater than zero and/or above thelimit of detection) indicates that the subject is in need of a treatmenttargeting EGFR (e.g., an anti-EGFR therapeutic agent).

In some embodiments, a level of EGFR expression that is higher than thenormal level of EGFR expression (e.g., from a subject that does not havea cancer) indicates that the subject has a cancer that is treatable witha treatment targeting EGFR (e.g., an anti-EGFR therapeutic agent). Insome embodiments, a level of EGFR expression that is 1.5×, 2×, 2.5×, 3×,3.5×, 4×, 4.5×, 5×, 6×, 7×, 8×, 9×, 10× or more than the normal level ofEGFR expression (e.g., from a subject that does not have a cancer)indicates that the subject has a cancer that is treatable with atreatment targeting EGFR (e.g., an anti-EGFR therapeutic agent).

In some embodiments, detecting expression of EGFRvIII indicates that thesubject has a cancer that is treatable with a treatment targeting EGFR(e.g., an anti-EGFR therapeutic agent). In some embodiments, detectingexpression of EGFRvIII (e.g., detecting expression greater than zeroand/or above the limit of detection) indicates that the subject has acancer that is treatable with a treatment targeting EGFR (e.g., ananti-EGFR therapeutic agent).

Therapies

In some embodiments, the technology is related to therapies, e.g.,cancer therapies. For example, in some embodiments a subject is testedfor cancer and then treated for cancer. In particular embodiments, EGFRexpression and/or EGFRvIII expression is assayed in a sample from asubject and the subject is treated with an anti-EGFR therapeutic agent.Particular anti-EGFR therapeutic agents include, but are not limited to,tyrosine kinase inhibitors (e.g., an adenosine triphosphate analog,e.g., an anilinoquinazoline (e.g., gefitinib, canertinib), lapatinib,erlotinib, etc.) and antibodies (e.g., monoclonal antibodies) such as,e.g., Cetuximab (Erbitux), Panitumumab, ABT-806, and ABT-414.

In some embodiments, the anti-EGFR therapeutic agent is Vandetanib(Caprelsa), Panitumumab (Vectibix), Gefitinib (Iressa), Erlotinib(Tarceva), or Afatinib (Gilotrif).

Some embodiments comprise treatment with a therapy targeting signalingdownstream of EGFR such as a therapy targeting the genes or theproduct(s) of genes (e.g., RNA and/or protein) encoding KRAS, BRAF, MEK,ERK, PI3K, phospholipase C gamma, AKT, and/or STAT.

Some embodiments provide a combination therapy comprising treating asubject with an anti-EGFR therapeutic agent and one or more of aradiation therapy or a chemotherapy (e.g., a platinum-based therapy, atopoisomerase inhibitor, a taxane). Some embodiments provide acombination therapy comprising treating a subject with more than oneanti-EGFR therapeutic agent. In some embodiments, combination therapiescomprise treating a subject with a dietary bioactive agent such ascapsaicin, genistein, or curcumin.

In some embodiments, the anti-EGFR therapy is an antibody thatrecognizes EGFR. The antibody can be a monoclonal antibody or apolyclonal antibody, and may be, for example, a human, humanized, orchimeric antibody. Monoclonal antibodies against target antigens areproduced by a variety of techniques including conventional monoclonalantibody methodologies such as the somatic cell hybridization techniquesof Köhler and Milstein (Nature, 256:495 (1975)). Although somatic cellhybridization procedures are preferred in some embodiments, othertechniques for producing monoclonal antibodies are contemplated as well(e.g., viral or oncogenic transformation of B lymphocytes).

In some embodiments, the antibody is ABT-806, which is a humanizedantibody specific for an epitope of EGFR that is exposed in deletionvariant EGFRvIII or when wild-type EGF receptors are amplified,overexpressed, or activated (see, e.g., Zhang et al. (2013), J Nucl Med54 (Supplement 2): 396).

In some embodiments, the antibody is ABT-414, which is an anti-EGFRmonoclonal antibody drug conjugate (see, e.g., Johns et al. (2002) Int JCancer 98(3): 398-408; Phillips et al. (2013) Mol Cancer Ther 12(11Suppl): Abstract A250). ABT-414 targets cancer cells by combining both achemotherapy drug (MMAF) with an antibody directed against the epidermalgrowth factor receptor (EGFR) (see, e.g., Jungbluth et al. (2003) ProcNatl Acad Sci USA 100(2): 639-44). This combination in a single drug iscalled an antibody drug conjugate (ADC). As an ADC, ABT-414 is stable inthe bloodstream and releases the potent chemotherapy agent only insidetargeted cancer cells. Studies are being conducted to determine if thisapproach can reduce the toxic side effects of traditional chemotherapywhile enhancing anti-tumor activity (see, e.g., Doronina et al. (2008)Bioconjug Chem 19: 1960-3).

In some embodiments, a subject is tested to assess the presence, theabsence, or the level of a disease (e.g., a cancer), e.g., bydetermining the presence of EGFRvIII expression (e.g., determining thepresence of EGFRvIII mRNA) and/or by quantifying total EGFR to determinethe risk of or the presence of cancer, and thereafter the subject istreated with an anti-cancer therapy (e.g., an anti-EGFR therapy) basedon the outcome of the test. In some embodiments, a patient is tested,treated, and then tested again to monitor the response to therapy. Insome embodiments, cycles of testing and treatment may occur withoutlimitation to the pattern of testing and treating (e.g., test/treat,test/treat/test, test/treat/test/treat, test/treat/test/treat/test,test/treat/treat/test/treat/treat, etc.), the periodicity, or theduration of the interval between each testing and treatment phase.

Samples

In some embodiments, nucleic acids (e.g., RNA) are isolated from abiological sample containing a variety of other components, such asproteins, lipids, and non-template nucleic acids. Nucleic acids can beobtained from any material (e.g., cellular material (live or dead),extracellular material, environmental samples (e.g., metagenomicsamples), synthetic material (e.g., amplicons such as provided by PCR orother amplification technologies)), tumor material, neoplastic material,etc., obtained from an animal Nucleic acid molecules can be obtaineddirectly from an organism or from a biological sample obtained from anorganism, e.g., from blood, urine, cerebrospinal fluid, seminal fluid,saliva, sputum, stool, hair, sweat, tears, skin, and tissue. Exemplarysamples include, but are not limited to, whole blood, lymphatic fluid,serum, plasma, buccal cells, sweat, tears, saliva, sputum, hair, skin,biopsy, cerebrospinal fluid (CSF), amniotic fluid, seminal fluid,vaginal excretions, serous fluid, synovial fluid, pericardial fluid,peritoneal fluid, pleural fluid, transudates, exudates, cystic fluid,bile, urine, gastric fluids, intestinal fluids, fecal samples, andswabs, aspirates (e.g., bone marrow, fine needle, etc.), washes (e.g.,oral, nasopharyngeal, bronchial, bronchialalveolar, optic, rectal,intestinal, vaginal, epidermal, etc.), and/or other specimens.

Any tissue or body fluid specimen may be used as a source for nucleicacid for use in the technology, including forensic specimens, archivedspecimens, preserved specimens, and/or specimens stored for long periodsof time, e.g., fresh-frozen, methanol/acetic acid fixed, orformalin-fixed paraffin embedded (FFPE) specimens and samples. Nucleicacid molecules can also be isolated from cultured cells, such as aprimary cell culture or a cell line. The cells or tissues from whichnucleic acids are obtained can be infected with a virus or otherintracellular pathogen. In particular embodiments, a sample is total RNAextracted from a biological specimen or a cDNA library. A sample mayalso be isolated RNA or cDNA from a non-cellular origin, e.g.amplified/isolated RNA or DNA that has been stored in a freezer.

Nucleic acid molecules can be obtained, e.g., by extraction from abiological sample, e.g., by a variety of techniques such as thosedescribed by Maniatis, et al. (1982) Molecular Cloning: A LaboratoryManual, Cold Spring Harbor, N.Y. (see, e.g., pp. 280-281).

In various embodiments, a nucleic acid is amplified. Any amplificationmethod known in the art may be used. Examples of amplificationtechniques that can be used include, but are not limited to, PCR,quantitative PCR, quantitative fluorescent PCR (QF-PCR), multiplexfluorescent PCR (MF-PCR), real time PCR, reverse transcription PCR(RT-PCR), single cell PCR, restriction fragment length polymorphism PCR(PCR-RFLP), hot start PCR, nested PCR, in situ polony PCR, in siturolling circle amplification (RCA), bridge PCR, picotiter PCR, andemulsion PCR. Other suitable amplification methods include the ligasechain reaction (LCR), transcription amplification, self-sustainedsequence replication, selective amplification of target polynucleotidesequences, consensus sequence primed polymerase chain reaction (CP-PCR),arbitrarily primed polymerase chain reaction (AP-PCR), degenerateoligonucleotide-primed PCR (DOP-PCR), and nucleic acid based sequenceamplification (NABSA). Other amplification methods that can be usedherein include those described in U.S. Pat. Nos. 5,242,794; 5,494,810;4,988,617; and 6,582,938.

Kits

Some embodiments of the technology relate to kits for detecting EGFRvIIIexpression (e.g., for detecting EGFRvIII mRNA or EGFRvIII cDNA) and/orfor detecting and/or quantifying total EGFR expression (e.g., fordetecting and/or quantifying total EGFR mRNA or cDNA produced from totalEGFR mRNA). Some embodiments of the technology relate to kits fordetecting EGFRvIII expression (e.g., for detecting EGFRvIII mRNA orEGFRvIII cDNA) and/or for detecting and/or quantifying total EGFRexpression (e.g., for detecting and/or quantifying total EGFR mRNA orcDNA produced from total EGFR mRNA) relative to the expression of acontrol mRNA such as, e.g., ACTB, ABL, or G6PD.

For instance, some embodiments provide a kit comprising a set of primersfor detecting EGFRvIII mRNA, e.g., an oligonucleotide comprising,consisting of, or consisting essentially of a sequence according to SEQID NO: 1 or SEQ ID NO: 31 (e.g., an oligonucleotide comprising,consisting of, or consisting essentially of a sequence or a reversecomplement of a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 1 or SEQ ID NO: 31 or that is 100% identical toSEQ ID NO: 1 or SEQ ID NO: 31) and an oligonucleotide comprising,consisting of, or consisting essentially of a sequence according to SEQID NO: 2 (e.g., an oligonucleotide comprising, consisting of, orconsisting essentially of a sequence or a reverse complement of asequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical toSEQ ID NO: 2 or that is 100% identical to SEQ ID NO: 2). In someembodiments, kits further comprise a probe for detecting EGFRvIII, e.g.,an oligonucleotide comprising, consisting of, or consisting essentiallyof a sequence according to SEQ ID NO: 3 (e.g., an oligonucleotidecomprising, consisting of, or consisting essentially of a sequence or areverse complement of a sequence that is at least 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to SEQ ID NO: 3 or that is 100% identical to SEQID NO: 3). In some embodiments, the oligonucleotide comprising,consisting of, or consisting essentially of a sequence according to SEQID NO: 3 comprises a detectable label (e.g., a fluorescent moiety). Insome embodiments, the oligonucleotide comprising, consisting of, orconsisting essentially of a sequence according to SEQ ID NO: 3 comprisesa detectable label (e.g., a fluorescent moiety) and a quencher.

Some embodiments of the technology relate to kits for detecting EGFRvIIIexpression (e.g., for detecting EGFRvIII mRNA or cDNA) and/or fordetecting and/or quantifying total EGFR expression (e.g., for detectingand/or quantifying total EGFR mRNA or cDNA produced from total EGFRmRNA). For instance, some embodiments provide a kit comprising a set ofprimers for detecting EGFRvIII mRNA, e.g., an oligonucleotidecomprising, consisting of, or consisting essentially of a sequenceaccording to SEQ ID NO: 1 or SEQ ID NO: 31 (e.g., an oligonucleotidecomprising, consisting of, or consisting essentially of a sequence or areverse complement of a sequence that is at least 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to SEQ ID NO: 1 or SEQ ID NO: 31 or that is 100%identical to SEQ ID NO: 1 or SEQ ID NO: 31) and an oligonucleotidecomprising, consisting of, or consisting essentially of a sequenceaccording to SEQ ID NO: 2 (e.g., an oligonucleotide comprising,consisting of, or consisting essentially of a sequence or a reversecomplement of a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 2 or that is 100% identical to SEQ ID NO: 2). Insome embodiments, kits further comprise a probe for detecting EGFRvIII,e.g., an oligonucleotide comprising, consisting of, or consistingessentially of a sequence according to SEQ ID NO: 3 (e.g., anoligonucleotide comprising, consisting of, or consisting essentially ofa sequence or a reverse complement of a sequence that is at least 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 3 or that is 100%identical to SEQ ID NO: 3). In some embodiments, the oligonucleotidecomprising, consisting of, or consisting essentially of a sequenceaccording to SEQ ID NO: 3 comprises a detectable label (e.g., afluorescent moiety). In some embodiments, the oligonucleotidecomprising, consisting of, or consisting essentially of a sequenceaccording to SEQ ID NO: 3 comprises a detectable label (e.g., afluorescent moiety) and a quencher. And, in some embodiments, kitsfurther comprise a set of primers for detecting and/or quantifying totalEGFR mRNA, e.g., an oligonucleotide comprising, consisting of, orconsisting essentially of a sequence according to SEQ ID NO: 7 (e.g., anoligonucleotide comprising, consisting of, or consisting essentially ofa sequence or a reverse complement of a sequence that is at least 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 7 or that is 100%identical to SEQ ID NO: 7) and an oligonucleotide comprising, consistingof, or consisting essentially of a sequence according to SEQ ID NO: 8(e.g., an oligonucleotide comprising, consisting of, or consistingessentially of a sequence or a reverse complement of a sequence that isat least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 8 orthat is 100% identical to SEQ ID NO: 8). In some embodiments, kitsfurther comprise a probe for detecting and/or quantifying total EGFR,e.g., an oligonucleotide comprising, consisting of, or consistingessentially of a sequence according to SEQ ID NO: 9 or SEQ ID NO: 32(e.g., an oligonucleotide comprising, consisting of, or consistingessentially of a sequence or a reverse complement of a sequence that isat least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 9 orSEQ ID NO: 32 or that is 100% identical to SEQ ID NO: 9 or SEQ ID NO:32). In some embodiments, the oligonucleotide comprising, consisting of,or consisting essentially of a sequence according to SEQ ID NO: 9 or SEQID NO: 32 comprises a detectable label (e.g., a fluorescent moiety). Insome embodiments, the oligonucleotide comprising, consisting of, orconsisting essentially of a sequence according to SEQ ID NO: 9 or SEQ IDNO: 32 comprises a detectable label (e.g., a fluorescent moiety) and aquencher.

Some embodiments of the technology relate to kits for detecting and/orquantifying total EGFR expression. In some embodiments, kits furthercomprise a set of primers for detecting and/or quantifying total EGFRmRNA, e.g., an oligonucleotide comprising, consisting of, or consistingessentially of a sequence according to SEQ ID NO: 7 (e.g., anoligonucleotide comprising, consisting of, or consisting essentially ofa sequence or a reverse complement of a sequence that is at least 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 7 or that is 100%identical to SEQ ID NO: 7) and an oligonucleotide comprising, consistingof, or consisting essentially of a sequence according to SEQ ID NO: 8(e.g., an oligonucleotide comprising, consisting of, or consistingessentially of a sequence or a reverse complement of a sequence that isat least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 8 orthat is 100% identical to SEQ ID NO: 8). In some embodiments, kitsfurther comprise a probe for detecting and/or quantifying total EGFR,e.g., an oligonucleotide comprising, consisting of, or consistingessentially of a sequence according to SEQ ID NO: 9 or SEQ ID NO: 32(e.g., an oligonucleotide comprising, consisting of, or consistingessentially of a sequence or a reverse complement of a sequence that isat least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 9 orSEQ ID NO: 32 or that is 100% identical to SEQ ID NO: 9 or SEQ ID NO:32). In some embodiments, the oligonucleotide comprising, consisting of,or consisting essentially of a sequence according to SEQ ID NO: 9 or SEQID NO: 32 comprises a detectable label (e.g., a fluorescent moiety). Insome embodiments, the oligonucleotide comprising, consisting of, orconsisting essentially of a sequence according to SEQ ID NO: 9 or SEQ IDNO: 32 comprises a detectable label (e.g., a fluorescent moiety) and aquencher.

Further embodiments of kits comprise a set of primers and a probe forthe detection and/or quantification of a control mRNA such as ACTB, ABL,or G6PD. Thus, in some embodiments kits comprise primers and a probe todetect ACTB, e.g., an oligonucleotide comprising, consisting of, orconsisting essentially of a sequence according to SEQ ID NO: 13 (e.g.,an oligonucleotide comprising, consisting of, or consisting essentiallyof a sequence or a reverse complement of a sequence that is at least80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 13 or that is100% identical to SEQ ID NO: 13), an oligonucleotide comprising,consisting of, or consisting essentially of a sequence according to SEQID NO: 14 (e.g., an oligonucleotide comprising, consisting of, orconsisting essentially of a sequence or a reverse complement of asequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical toSEQ ID NO: 14 or that is 100% identical to SEQ ID NO: 14), and a probecomprising an oligonucleotide sequence according to SEQ ID NO: 15 (e.g.,an oligonucleotide comprising, consisting of, or consisting essentiallyof a sequence or a reverse complement of a sequence that is at least80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 15 or that is100% identical to SEQ ID NO: 15). In some embodiments, kits compriseprimers and a probe to detect ABL, e.g., an oligonucleotide comprising,consisting of, or consisting essentially of a sequence according to SEQID NO: 19 (e.g., an oligonucleotide comprising, consisting of, orconsisting essentially of a sequence or a reverse complement of asequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical toSEQ ID NO: 19 or that is 100% identical to SEQ ID NO: 19), anoligonucleotide comprising, consisting of, or consisting essentially ofa sequence according to SEQ ID NO: 20 (e.g., an oligonucleotidecomprising, consisting of, or consisting essentially of a sequence or areverse complement of a sequence that is at least 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to SEQ ID NO: 20 or that is 100% identical to SEQID NO: 20), and a probe comprising an oligonucleotide sequence accordingto SEQ ID NO: 21 (e.g., an oligonucleotide comprising, consisting of, orconsisting essentially of a sequence or a reverse complement of asequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical toSEQ ID NO: 21 or that is 100% identical to SEQ ID NO: 21). In someembodiments, kits comprise primers and a probe to detect G6PD, e.g., anoligonucleotide comprising, consisting of, or consisting essentially ofa sequence according to SEQ ID NO: 22 (e.g., an oligonucleotidecomprising, consisting of, or consisting essentially of a sequence or areverse complement of a sequence that is at least 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to SEQ ID NO: 22 or that is 100% identical to SEQID NO: 22), an oligonucleotide comprising, consisting of, or consistingessentially of a sequence according to SEQ ID NO: 23 (e.g., anoligonucleotide comprising, consisting of, or consisting essentially ofa sequence or a reverse complement of a sequence that is at least 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 23 or that is 100%identical to SEQ ID NO: 23), and a probe comprising an oligonucleotidesequence according to SEQ ID NO: 24 (e.g., an oligonucleotidecomprising, consisting of, or consisting essentially of a sequence or areverse complement of a sequence that is at least 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to SEQ ID NO: 24 or that is 100% identical to SEQID NO: 24). In some embodiments, the oligonucleotide comprising,consisting of, or consisting essentially of a sequence according to SEQID NO: 15, SEQ ID NO: 21, and/or SEQ ID NO: 24 comprises a detectablelabel (e.g., a fluorescent moiety). In some embodiments, theoligonucleotide comprising, consisting of, or consisting essentially ofa sequence according to SEQ ID NO: 15, SEQ ID NO: 21, and/or SEQ ID NO:24 comprises a detectable label (e.g., a fluorescent moiety) and aquencher.

Computer-Aided Diagnostics

In some embodiments, a computer-based analysis program is used totranslate the raw data generated by the detection assay (e.g., raw datacomprising an EGFRvIII Ct value, a total EGFR Ct value, a EGFRvIII dCtvalue, a total EGFR dCt value; e.g., raw data comprising an indicationof the presence, absence, or the amount of EGFRvIII and/or the amount oftotal EGFR expression) into data of predictive value for a clinician.The clinician can access the predictive data using any suitable means.Thus, in some preferred embodiments, the present technology provides thefurther benefit that the clinician, who is not likely to be trained ingenetics or molecular biology, need not understand the raw data. Thedata is presented directly to the clinician in its most useful form. Theclinician is then able to utilize the information to optimize the careof the subject. The present technology contemplates any method capableof receiving, processing, and transmitting the information to and fromlaboratories conducting the assays, information providers, medicalpersonnel, and subjects. For example, in some embodiments of the presenttechnology, a sample is obtained from a subject and submitted to aprofiling service (e.g., a clinical lab at a medical facility, genomicprofiling business, etc.), located in any part of the world (e.g., in acountry different than the country where the subject resides or wherethe information is ultimately used) to generate raw data. Where thesample comprises a tissue or other biological sample, the subject mayvisit a medical center to have the sample obtained and sent to theprofiling center, or subjects may collect the sample themselves anddirectly send it to a profiling center. Where the sample comprisespreviously determined biological information, the information may bedirectly sent to the profiling service by the subject. Once received bythe profiling service, the sample is processed and a profile is producedthat is specific for the diagnostic or prognostic information desiredfor the subject. The profile data is then prepared in a format suitablefor interpretation by a treating clinician. For example, rather thanproviding raw data, the prepared format may represent a diagnosis (e.g.,of a cancer) or risk assessment (e.g., of a cancer) for the subject,along with recommendations for particular treatment options. The datamay be displayed to the clinician by any suitable method. For example,in some embodiments, the profiling service generates a report that isprinted for the clinician (e.g., at the point of care) or displayed tothe clinician on a computer monitor. In some embodiments, theinformation is first analyzed at the point of care or at a regionalfacility. The raw data is then sent to a central processing facility forfurther analysis and/or to convert the raw data into information usefulfor a clinician or patient. The central processing facility provides theadvantage of privacy (e.g., all data is stored in a central facilitywith uniform security protocols), speed, and uniformity of dataanalysis. The central processing facility can then control the fate ofthe data following treatment of the subject. For example, using anelectronic communication system, the central facility can provide datato the clinician, the subject, or researchers. In some embodiments, thesubject is able to access the data directly using the electroniccommunication system. The subject may chose further intervention orcounseling based on the results. In some embodiments, the data is usedfor research use. For example, the data may be used to optimize theinclusion or elimination of markers as useful indicators of a particularcondition associated with the disease.

Thus, in some embodiments, the technology described herein is associatedwith a programmable machine designed to perform a sequence of arithmeticor logical operations as provided by the methods described herein. Forexample, some embodiments of the technology are associated with (e.g.,implemented in) computer software and/or computer hardware. In oneaspect, the technology relates to a computer comprising a form ofmemory, an element for performing arithmetic and logical operations, anda processing element (e.g., a microprocessor) for executing a series ofinstructions (e.g., a method as provided herein) to read, manipulate,and store data. In some embodiments, a microprocessor is part of asystem for determining the absence, presence, and/or amount of EGFRvIIIand/or total EGFR expression (e.g., relative to an internal control);generating standard curves; determining a Ct value; calculating a dCtvalue, e.g., as described herein or as is known in the art.

In some embodiments, a microprocessor or computer uses the absence,presence, and/or amount of EGFRvIII and/or total EGFR expression (e.g.,relative to an internal control) in an algorithm to predict a site of acancer.

In some embodiments, a software or hardware component receives theresults of multiple assays and determines a single value result toreport to a user that indicates a cancer risk based on the results ofthe multiple assays (e.g., determining the absence, presence, and/oramount of EGFRvIII and/or total EGFR expression (e.g., relative to aninternal control)). Related embodiments calculate a risk factor based ona mathematical combination (e.g., a weighted combination, a linearcombination) of the results from multiple assays, e.g., determining theabsence, presence, and/or amount of EGFRvIII and/or total EGFRexpression (e.g., relative to an internal control). In some embodiments,the absence, presence, and/or amount of EGFRvIII and/or total EGFRexpression (e.g., relative to an internal control) defines a dimensionand may have values in a multidimensional space and the coordinatedefined by the absence, presence, and/or amount of EGFRvIII and/or totalEGFR expression (e.g., relative to an internal control) is a result,e.g., to report to a user, e.g., related to a cancer risk.

Some embodiments comprise a storage medium and memory components. Memorycomponents (e.g., volatile and/or nonvolatile memory) find use instoring instructions (e.g., an embodiment of a process as providedherein) and/or data (e.g., a work piece such as the absence, presence,and/or amount of EGFRvIII and/or total EGFR expression (e.g., relativeto an internal control), sequences, and statistical descriptionsassociated therewith). Some embodiments relate to systems alsocomprising one or more of a CPU, a graphics card, and a user interface(e.g., comprising an output device such as display and an input devicesuch as a keyboard).

Programmable machines associated with the technology compriseconventional extant technologies and technologies in development or yetto be developed (e.g., a quantum computer, a chemical computer, a DNAcomputer, an optical computer, a spintronics based computer, etc.).

In some embodiments, the technology comprises a wired (e.g., metalliccable, fiber optic) or wireless transmission medium for transmittingdata. For example, some embodiments relate to data transmission over anetwork (e.g., a local area network (LAN), a wide area network (WAN), anad-hoc network, the internet, etc.). In some embodiments, programmablemachines are present on such a network as peers and in some embodimentsthe programmable machines have a client/server relationship.

In some embodiments, data are stored on a computer-readable storagemedium such as a hard disk, flash memory, optical media, a floppy disk,etc.

In some embodiments, the technology provided herein is associated with aplurality of programmable devices that operate in concert to perform amethod as described herein. For example, in some embodiments, aplurality of computers (e.g., connected by a network) may work inparallel to collect and process data, e.g., in an implementation ofcluster computing or grid computing or some other distributed computerarchitecture that relies on complete computers (with onboard CPUs,storage, power supplies, network interfaces, etc.) connected to anetwork (private, public, or the internet) by a conventional networkinterface, such as Ethernet, fiber optic, or by a wireless networktechnology.

For example, some embodiments provide a computer that includes acomputer-readable medium. The embodiment includes a random access memory(RAM) coupled to a processor. The processor executes computer-executableprogram instructions stored in memory. Such processors may include amicroprocessor, an ASIC, a state machine, or other processor, and can beany of a number of computer processors, such as processors from IntelCorporation of Santa Clara, Calif. and Motorola Corporation ofSchaumburg, Ill. Such processors include, or may be in communicationwith, media, for example computer-readable media, which storesinstructions that, when executed by the processor, cause the processorto perform the steps described herein.

Embodiments of computer-readable media include, but are not limited to,an electronic, optical, magnetic, or other storage or transmissiondevice capable of providing a processor with computer-readableinstructions. Other examples of suitable media include, but are notlimited to, a floppy disk, CD-ROM, DVD, magnetic disk, memory chip, ROM,RAM, an ASIC, a configured processor, all optical media, all magnetictape or other magnetic media, or any other medium from which a computerprocessor can read instructions. Also, various other forms ofcomputer-readable media may transmit or carry instructions to acomputer, including a router, private or public network, or othertransmission device or channel, both wired and wireless. Theinstructions may comprise code from any suitable computer-programminglanguage, including, for example, Swift, Objective C, C, C++, C#, VisualBasic, Java, Python, Perl, Ruby, Unix, and JavaScript.

Computers are connected in some embodiments to a network. Computers mayalso include a number of external or internal devices such as a mouse, aCD-ROM, DVD, a keyboard, a display, or other input or output devices.Examples of computers are personal computers, digital assistants,personal digital assistants, cellular phones, mobile phones, smartphones, pagers, digital tablets, laptop computers, internet appliances,and other processor-based devices. In general, the computers related toaspects of the technology provided herein may be any type ofprocessor-based platform that operates on any operating system, such asMicrosoft Windows, Linux, UNIX, Mac OS X, BSD, etc., capable ofsupporting one or more programs comprising the technology providedherein. Some embodiments comprise a personal computer executing otherapplication programs (e.g., applications). The applications can becontained in memory and can include, for example, a word processingapplication, a spreadsheet application, an email application, an instantmessenger application, a presentation application, an Internet browserapplication, a calendar/organizer application, and any other applicationcapable of being executed by a client device.

All such components, computers, and systems described herein asassociated with the technology may be logical or virtual.

EXAMPLES Example 1—Detection of Total EGFR mRNA and EGFRvIII mRNA inCancer Samples

During the development of embodiments of the technology provided herein,experiments were conducted to test the detection of EGFRvIII mRNA andtotal EGFR mRNA in formalin-fixed, paraffin embedded (FFPE) glioblastomamyeloma (GBM) samples using novel primers and probes as describedherein.

Methods

Preparation of RNA (FFPE) GBM specimens: Paraffin embedded (FFPE)glioblastoma myeloma (GBM) samples were processed from previous Phase Itrials of the ABT-806 and ABT-414 therapeutics. One FFPE section wasprepared from each of 21 GBM specimens examined in the experiments. EachFFPE section was scraped into a microfuge tube using a single-edge razorblade. A new blade was used for each sample. A FFPE section from a lungsample (“A549”) known to express EGFR was scraped into a microfuge tubeas a positive control (e.g., available at ATCC® CCL-185™). See, e.g.,Giard et al (1973) “In vitro cultivation of human tumors: establishmentof cell lines derived from a series of solid tumors” J. Natl. CancerInst. 51: 1417-1423.

The samples were identified by the following sample names: 1250, 1238,1237, 1236, 1248, 793, 783, 782, 779, 1247, 630, 1246, 1242, 1243, 401,402, 403, 302, 1251, 1252, and 1253.

The FFPE sections were processed for RNA extraction using the QiagenRNeasy FFPE extraction kit according to the manufacturer's protocol(e.g., RNeasy FFPE Handbook, Qiagen). Deparaffinization solution (QiagenRNeasy FFPE extraction kit) alone (without added FFPE tissue) was usedas a negative control.

RNA was extracted from samples by adding 160 microliters ofdeparaffinization solution (Qiagen RNeasy FFPE extraction kit) to thetubes containing the FFPE material. The samples were vortexed vigorouslyfor 10 seconds and then centrifuged briefly to collect the sample at thebottom of the tube. Next, the samples were incubated at 56° C. for 3minutes and then allowed to cool at room temperature. A 150-microlitervolume of Buffer PKD (Qiagen RNeasy FFPE extraction kit) was added, thesamples were mixed by vortexing, and then the samples were centrifugedfor 1 minute at 11,000×g. After centrifugation, 10 microliters ofproteinase K (Qiagen RNeasy FFPE extraction kit) were added to thelower, clear phase and the samples were mixed gently by pipetting up anddown. Then, samples were first incubated at 56° C. for 15 minutes, andthen samples were incubated at 80° C. for 15 minutes. The lower,uncolored phase of each sample was transferred into a new 2-milliletermicrocentrifuge tube and incubated on ice for 3 minutes. The sampleswere then centrifuged for 15 minutes at 20,000×g. After centrifugation,the supernatant was transferred to a new microcentrifuge tube withoutdisturbing the pellet.

Next, 15 microliters of DNase Booster Buffer (Qiagen RNeasy FFPEextraction kit) and 10 microliters of RNase-free DNase I stock solution(Qiagen RNeasy FFPE extraction kit) were added to the supernatant andthe samples were mixed by inverting the tubes. The samples were thencentrifuged to collect the contents at the bottoms of the tubes; sampleswere incubated at room temperature for 15 minutes. After incubation onice, 320 microliters of Buffer RBC (Qiagen RNeasy FFPE extraction kit)were added to adjust binding conditions and the lysates were mixedthoroughly. A 720-microliter volume of ethanol (100%) was added to eachsample and the samples were mixed well by pipetting. After mixing, 700microliters of the sample were transferred (including any precipitatethat may have formed) to an RNeasy MinElute spin column (Qiagen RNeasyFFPE extraction kit) placed in a 2-milliliter collection tube. The lidwas closed gently and the tubes were centrifuged for 15 seconds at8000×g or more (greater than 10,000 rpm). The flow-through wasdiscarded. The centrifugation was repeated until the entire sample hadpassed through the RNeasy MinElute spin column. Next, 500 microliters ofBuffer RPE (Qiagen RNeasy FFPE extraction kit) was added to the RNeasyMinElute spin column. The lid was closed gently and the samples werecentrifuged for 15 seconds at 8000×g or more (greater than 10,000 rpm).The flow-through was discarded. Another volume of 500 microliters ofBuffer RPE (Qiagen RNeasy FFPE extraction kit) were added to the RNeasyMinElute spin column. The lid was closed gently and the samples werecentrifuged for 2 minutes at 8000×g or more (greater than 10,000 rpm) towash the spin column membrane. The flow-through was discarded.

After centrifugation, each RNeasy MinElute spin column was carefullyremoved from the collection tube and placed in a new 2-millilitercollection tube. The lid of each spin column was opened and the tubeswere centrifuged at full speed for 5 minutes. The collection tubesholding the flow-through were discarded. The RNeasy MinElute spin columnwas next placed in a new 1.5-millileter collection tube. A volume of 30microliters of RNase-free water was added directly to the spin columnmembrane. The lid was closed gently and the sample was centrifuged for 1minute at full speed to elute and collect the RNA.

Testing GBM samples using EGFRvIII assay: Two reaction mixtures (e.g.,Master Mixes) were prepared—Master Mix A comprised the nominal EGFRvIIIreverse primer EGvIII-A and Master Mix B comprised a shorter,alternative EGFRvIII reverse primer, EGFRvIII Rev_D designed for testingin embodiments of methods described herein. First, a Common Master Mix(CMM) was prepared and then Master Mix A and Master Mix B were preparedusing the CMM (see, e.g., TABLE 6).

The CMM comprised primers and probes for detecting total EGFR (e.g., theprimers EGwti29_−47 to −25 and EGwti29_+23 to −3 and the probe EGi29_−24to −11pNED) and comprised the forward primer and probe for detectingEGFRvIII (e.g., the primer EGvIIIi1_−58 to −41 and the probeEGvIIIi1_−39 to −21 pFAM) when paired with one of the reverse primers inthe Master Mix A or Master Mix B (EGvIII-A and EGFRvIII Rev_D,respectively). See FIG. 2 for locations of total EGFR primers and probeand EGFRvIII primers and probe.

TABLE 6 Exemplary amplification reaction mixtures component stockcomponent in total rxn volume number component solution reaction volume(μL) of final volume component conc. unit final Conc. unit (μL) per rxnrxns (μL) Common Master Mix (CMM) 5 × EZ Buffer 5 × 1.000 × 50 10.000104 1040.00 dNTP Mix 25 mM 0.488 mM 50 0.975 104 101.40 ROX Dye 2 μM0.029 μM 50 0.725 104 75.40 Aptamer 24.82 μM 0.200 μM 50 0.403 104 41.90EGwti29_−47 to −25 50.79 μM 0.050 μM 50 0.049 104 5.12 EGi29_−24 to−11pNED 42.85 μM 0.600 μM 50 0.700 104 72.81 EGwti29_+23 to −3 55.17 μM0.075 μM 50 0.068 104 7.07 EGvIIIi1_−58 to −41 55.34 μM 0.063 μM 500.056 104 5.87 EGvIIIi1_−39 to −21pFAM 32.22 μM 0.188 μM 50 0.291 10430.26 bActI1_−53 to −33 53.28 μM 0.100 μM 50 0.094 104 9.76 bActI1_+17to −5 53.24 μM 0.300 μM 50 0.282 104 29.30 bActI1_−29 to −10pCY5 52.86μM 0.300 μM 50 0.284 104 29.51 MnCl₂ 30 mM 3.000 mM 50 5.000 104 520.00rTth 3.2 U/μL 0.200 U/μL 50 3.125 104 325.00 H₂O N/A N/A N/A N/A 500.948 104 98.59 Total 2392.00 Master Mix A CMM N/A N/A N/A N/A 50 23.00051 1173.00 EGvIII-A 53.44 μM 0.188 μM 50 0.175 51 8.95 H₂O N/A N/A N/AN/A 50 1.825 51 93.05 Master Mix B CMM N/A N/A N/A N/A 50 23.000 511173.00 EGFRvIII Rev_D 56.89 μM 0.423 μM 50 0.372 51 18.96 H₂O N/A N/AN/A N/A 50 1.628 51 83.04

In TABLE 6, probes with names ending in “NED”, “FAM”, and “CY5”comprised a NED, FAM, and CY5 fluorescent moiety, respectively.

The RNA eluates prepared above (GBM specimens), the positive EGFRcontrol (A549), and the negative control were diluted in water (e.g., 2microliters of sample in a 25 microliter volume). The 25-microlitersamples were added to 25 microliters of a Master Mix in each well of a96-well plate. Two replicates of each sample were tested. In addition,two replicates of a circular EGFR control plasmid (see below) were addedas a PCR positive control. The plate was sealed with an optical adhesivecover, centrifuged at 3000 rpm for 1 minute, placed in an ABI 7500 RtPCRSystem (m2000rt), and thermocycled according to the following program:

Stage Cycles Step Temp (C.) Time (min:sec) 1 1 1 62 30:00 2 4 1 92 00:302 60 00:30 (1-s autoincrement) 3 50 1 92 00:30 2 62 00:30 (1-sautoincrement) 3 58 (read*) 00:40 *in the “read” steps, the fluors FAM,NED, Cy5, and ROX are monitored.

Data were analyzed using software for generating and analyzing real-timePCR data (e.g., plotting data, data reduction, determining Ct values,etc.). Threshold values were FAM=0.100, NED=0.075, and Cy5=0.100.Results were tabulated and analyzed using Microsoft Excel.

Concentration determination for GBM specimens: The RNA concentrations ofGBM samples were determined by measuring absorbance of the samples at260 nanometers (using a Nanodrop spectrophotometer) and measuringabsorbance of the samples at 280 nanometers, then computing the ratio ofthe absorbance at 260 nanometers to the absorbance at 280 nanometers(A260/A280). For all samples, 2 microliters of eluate were added to theNanoDrop in replicates of 2. The concentrations were averaged toestimate the final concentration of RNA in nanograms per microliter(ng/μl).

Results

Real-time PCR data were collected and Ct values were determined fortotal EGFR mRNA, EGFRvIII mRNA, and the endogenous control ACTB mRNA.Data were collected from assays of reaction mixtures comprising thenominal EGFRvIII reverse primer EGvIII-A (see, e.g., FIG. 5, FIG. 7) andfrom assays of reaction mixtures comprising the alternate EGFRvIIIreverse primer EGFRvIII Rev_D tested herein (see, e.g., FIG. 6, FIG. 8).

Delta Ct values (dCt) for total EGFR mRNA and EGFRvIII mRNA werecalculated by subtracting the total EGFR mRNA Ct value from theendogenous control ACTB mRNA Ct value (e.g., “actin—EGFR”) and bysubtracting the EGFRvIII mRNA Ct value from the endogenous control ACTBmRNA Ct value (e.g., “actin—EGFRvIII), respectively (see, e.g., FIG. 5,FIG. 6, FIG. 7, FIG. 8). Accordingly, the dCt values provide a relativemeasure of gene expression using the expression of the endogenouscontrol ACTB mRNA as a baseline value.

The dCt values for the amounts of total EGFR mRNA in the GBM samplesranged from approximately 0 (zero), indicating “high” expression, toapproximately −7 or −8 (negative 7 or negative 8), indicating “lowexpression” (see, e.g., FIG. 5, FIG. 7). The data collected fromexperiments measuring total EGFR mRNA in the presence of the nominalEGFRvIII reverse primer EGvIII-A (see, e.g., FIG. 5) and fromexperiments measuring total EGFR mRNA in the presence of the alternateEGFRvIII reverse primer EGFRvIII Rev_D (see, e.g., FIG. 7A) were similar(see, e.g., FIG. 7B).

Expression of EGFRvIII mRNA was detected in five of the samples (GBM1248, GBM 1247, GBM 1246, GBM 1242, and GBM 401; see, e.g., asterisked(“*”) data in FIG. 5). Expression of EGFRvIII mRNA in these samplesranged from approximately −2 or −3 (negative 2 or negative 3) toapproximately −6 or −7 (negative 6 or negative 7) (see, e.g., FIG. 6).

In sum, the real-time EGFRvIII assay indicated that 5 of the 21 sampleswere positive for EGFRvIII mRNA. The relative total EGFR expression(actin Ct—EGFR Ct) ranged from 0.00 (high expression) to −7.34 (lowexpression). Total RNA yields for all tested GBM samples ranged from 6.3ng/μl to 237.3 ng/μl.

For the five samples in which expression of EGFRvIII mRNA was detectedusing the nominal reverse primer (see, e.g., FIG. 5, FIG. 6), similarexpression of EGFRvIII mRNA was detected using the novel, alternatereverse primer EGFRvIII Rev_D developed for the assays described herein(see, e.g., FIG. 8B, samples GBM 1248, GBM 1247, GBM 1246, GBM 1242, andGBM 401).

In addition, several more specimens were detected to have low-levelEGFRvIII signals using the alternative EGFRvIII reverse primer EGFRvIIIRev_D (see, e.g., FIG. 8). However, due to the high Ct and inefficientreaction kinetics (e.g., as determined by the relative reactionefficiency value MR) for these particular sample assays, it appearedthat these data were non-specific and would therefore be screened out byother filters.

Finally, experiments were conducted in which the concentration of totalRNA extracted from each GBM sample was correlated with the endogenouscontrol ACTB mRNA Ct value to assess the endogenous control ACTB mRNA Ctvalue as an indication of total RNA concentration in the samples (see,e.g., FIG. 9). The mean Ct from duplicate determinations of ACTB Ct wereplotted against the log₂ of the measured total RNA concentration (e.g.,by NanoDrop analysis of A₂₆₀/A₂₈₀) (see, e.g., FIG. 9). The dataindicated an approximately linear relationship between the ACTB Ct andthe log₂ of the RNA concentration (see, e.g., FIG. 9).

Example 2—Positive Controls

During the development of embodiments of the technology provided herein,experiments were conducted to assess the linear response of real-timePCR assays of a positive control plasmid comprising EGFRvIII, EGFR, andACTB nucleotide sequences. In particular, a control plasmid (pJW02) wasconstructed to comprise the EGFRvIII exon 1/exon 2 junction, the totalEGFR exon 29/exon 30 junction, and the ACTB exon 1/exon 2 junction (see,e.g., FIG. 10).

Methods

A reaction mixture (Master Mix) was prepared according to TABLE 7:

TABLE 7 Positive control Master Mix component stock component in totalrxn volume number component solution reaction volume (μL) of finalvolume component conc. unit final conc. unit (μL) per rxn rxns (μL) 5 ×EZ Buffer 5 × 1.000 × 50 10.000 105 1050.00 dNTP Mix 25 mM 0.488 mM 500.975 105 102.38 Rox Dye 2 μM 0.029 μM 50 0.725 105 76.13 Aptamer 24.82μM 0.200 μM 50 0.403 105 42.30 EGwti29_−47 to −25 50.79 μM 0.050 μM 500.049 105 5.17 EGwti29_+23 to −3 55.17 μM 0.075 μM 50 0.068 105 7.14EGi29_−24 to −11pNED 49.83 μM 0.600 μM 50 0.602 105 63.21 EGvIIIi1_−58to −41 55.34 μM 0.063 μM 50 0.056 105 5.93 EGvIII-A 53.44 μM 0.188 μM 500.175 105 18.42 EGvIIIi1_−39 to −21pFAM 32.22 μM 0.188 μM 50 0.291 10530.55 bActI1_−53 to −33 53.28 μM 0.100 μM 50 0.094 105 9.85 bActI1_+17to −5 53.24 μM 0.300 μM 50 0.282 105 29.58 bActI1_−29 to −10pCY5 55.95μM 0.300 μM 50 0.268 105 28.15 MnCl₂ 30 mM 3.000 mM 50 5.000 105 525.00rTth 3.2 U/μL 0.200 U/μL 50 3.125 105 328.13 H₂O N/A N/A N/A N/A 502.886 105 303.06 Total 2625

Circular pJW02 plasmid was serially diluted in 2-fold increments from 10nanograms per reaction to approximately 1.86×10⁻⁸ nanograms per reactionto create 31 dilution levels. Plasmid copy number ranged from over 1×10⁹copies per reaction to approximately 1 copy per reaction. 25 microlitersof template was added to 25 microliters of Master Mix in each well of a96-well plate. Three independent replicates of each dilution level weretested. Three replicates of H₂O were also tested as a negative (“notarget”) control. The plate was sealed with an optical adhesive cover,centrifuged at 3000 rpm for 1 minute, placed in an ABI 7500 RtPCRSystem, and thermocycled according to the following program:

Stage Cycles Step Temp (C.) Time (min:sec) 1 1 1 62 30:00 2 4 1 92 00:302 60 00:30 (1-s autoincrement) 3 50 1 92 00:30 2 62 00:30 (1-sautoincrement) 3 58 (read*) 00:40 *in the “read” steps, the fluors FAM,NED, Cy5, and ROX are monitored.

Data were analyzed using software for generating and analyzing real-timePCR data (e.g., plotting data, data reduction, determining Ct values,etc.). Threshold values for FAM=0.100, NED=0.075, and Cy5=0.100. Resultswere tabulated and analyzed in Microsoft Excel.

Results

The data collected indicated that the positive control plasmid (pJW02)provides adequate linearity for all three targets (EGFRvIII, total EGFR,and ACTB; see, e.g., FIG. 11, diamonds, squares, and triangles,respectively, indicating EGFRvIII, total EGFR, and ACTB mRNA) across awide range of Ct values (see, e.g., FIG. 11). The linear range is over7.5 logs (e.g., from 1.00 log₁₀ to −6.58 log₁₀), which corresponds to arange of 10 nanograms to 0.3 femtograms of plasmid per reaction. Datapoints from samples that provided less than 100% detection for all threetargets were discarded (open symbols, FIG. 11). These samples allcomprised the most dilute concentrations of RNA.

Example 3—Comparison of Internal Controls

During the development of embodiments of the technology provided herein,experiments were conducted to evaluate internal controls for use inembodiments of the total EGFR and EGFRvIII assays described herein. Inparticular, mRNAs for three internal controls were selected for thisstudy: beta-actin (ACTB), c-Abl 1 (ABL), and glucose 6 phosphatedehydrogenase (G6PD) (see, e.g., FIG. 12). ACTB resides on chromosome 7with EGFR; in contrast, neither ABL nor G6PD resides on chromosome 7.Tests were conducted to compare the performance of the internal controlsfor the EGFR assay and to evaluate the use of an internal control thatresides on the same chromosome as EGFR. It was contemplated that theACTB control would have higher expression levels in tumors withchromosome 7 duplications, but not in tumors with EGFR focalamplification. The ACTB mRNA sequence is available at NCBI accessionnumber NM_001101; the ABL mRNA sequence is available at NCBI accessionnumber NM_005157; and the G6PD mRNA sequence is available at NCBIaccession number NM_001042351, each incorporated by reference herein.

Methods

Three amplification Master Mixes were made, each of which compriseddNTP, aptamer, buffer, MnCl₂, recombinant Tth polymerase, ROX passivereference dye, the EGFR wild-type primers and probes in TABLE 8, and theEGFRvIII primers and probes in TABLE 7.

TABLE 8 Primers and probes used in the Master Mix oligo name oligo typestock concentration (μM) EGvIIIi1_−58to−41 primer 55.75 EGvIII-A primer53.61 EGvIIIi1_−39to−21pFAM probe 54.92 EGwti7_−28to−9 primer 53.48EGy3i1_+36to+18 primer 52.95 EGFRwti7_−7to+8 NED MGB probe 50.29bActl1_−53to−33 primer 52.58 bActl1_−29to−10pCY5 probe 52.34bActl1_+17to-5 primer 54.62 ABLi3_fwd−21to+2 primer 50.40 ABLi3+65to+41primer 43.84 ABLi3+9to+33pCY5 probe 36.76 G6PDl2_FWD−50TO−28 primer54.45 G6PDi2_rev−37to−12 primer 45.82 G6PDv1_fwd346to367 Cy5 probe 53.55

Primer and probe binding regions for ACTB are indicated in FIG. 13;primer and probe binding regions for ABL are indicated in FIG. 14; andprimer and probe binding regions are indicated in FIG. 15.

TABLE 9 FFPE Master Mix component stock component in total rxn volumenumber component solution reaction volume (μL) of final volume componentconc. unit final conc. unit (μL) per rxn rxns (μL) 5 × EZ Buffer 5 ×1.000 × 50 10.000 120 1200.00 dNTP Mix 25 mM 0.325 mM 50 0.650 120 78.00ROX Dye 2 μM 0.015 μM 50 0.368 120 44.10 Aptamer 24.82 μM 0.200 μM 500.403 120 48.35 EGvIIIi1_−58 to −41 55.75 μM 0.050 μM 50 0.045 120 5.38EGvIIIi1_−39 to −21pFAM 38.44 μM 0.150 μM 50 0.195 120 23.41 EGwti7_−28to −9 53.48 μM 0.050 μM 50 0.047 120 5.61 EGv3i1_+36 to +18 52.95 μM0.600 μM 50 0.567 120 67.99 EGFRwti7_−7 to +8 NED 49.92 μM 0.450 μM 500.451 120 54.09 MGB MnCl₂ Activation 100 mM 3.000 mM 50 1.500 120 180.00rTth 3.2 U/μL 0.200 U/μL 50 3.125 120 375.00 H₂O N/A N/A N/A N/A 502.651 120 318.07

TABLE 10 ACTB, ABL, and G6PD primer and probe mixes stock final totalrxn total conc. conc. volume component number component mix component(μM) (μM) (μL) volume (μL) of rxns volume (μL) name bActI1_−53 to −3352.58 0.050 50 0.048 40 1.902 ACTB bActI1_−29 to −10pCY5 52.34 0.150 500.143 40 5.732 bActI1_+17 to −5 54.62 0.150 50 0.137 40 5.492 H2O NA NA50 4.672 40 186.874 ABLi3_fwd−21 to +2 50.40 0.050 50 0.050 40 1.984 ABLABLi3 +65 to +41 43.84 0.150 50 0.171 40 6.844 ABLi3 +9 to +33pCY5 36.760.150 50 0.204 40 8.160 H2O NA NA 50 4.575 40 183.012 G6PDI2_fwd−50 to−28 54.45 0.050 50 0.046 40 1.836 G6PD G6PDi2_rev−37 to −12 45.82 0.15050 0.164 40 6.547 G6PDv1_fwd346 to 367pCy5 53.55 0.150 50 0.140 40 5.603H2O NA NA 50 4.650 40 186.014

TABLE 11 Internal control reaction mixes beta-actin rxn ABL rxn G6PD rxnMaster Mix (μL) 784.00 784.00 784.00 ACTB mix (μL) 196.00 ABL mix (μL)196.00 G6PD mix (μL) 196.00

An aliquot of the FFPE Master Mix (Table 9) was mixed with aliquots fromone of the three primer and probe mixes (Table 10) according to thevolumes in Table 11 to produce three internal control reaction mixescomprising one of the three internal control primer and probe sets(e.g., for ACTB, ABL, or G6PD). Target RNAs used were the A549 EGFR mRNApositive control and a positive control known to express EGFRvIII mRNA(“U87vIII”), which was isolated from a GBM FFPE sample and confirmed toexpress EGFRvIII mRNA. U87vIII was produced by stably transfecting aglioblastoma-derived cell line (U87-MG) with the EGFRvIII mutant.10-fold serial dilutions were made of each RNA in H₂O to provide a rangeof from 50 nanograms of RNA to 0.0005 nanograms (0.5 picograms) of RNAper reaction. 50 nanograms of genomic DNA and water were used fornegative controls. 25 μL of Master Mix and 25 μL of target RNA wereadded to each well of a 96-well plate and thermocycled according to thefollowing program:

Stage Cycles Step Temp (C.) Time (min:sec) 1 1 1 62 30:00 2 4 1 92 00:302 60 00:30 (1-s autoincrement) 3 56 1 92 00:30 2 62 00:30 (1-sautoincrement) 3 58 (read*) 00:40 *in the “read” steps, the fluors FAM,NED, Cy5, and ROX are monitored.

Data were analyzed using software for generating and analyzing real-timePCR data (e.g., plotting data, data reduction, determining Ct values,etc.). Threshold values for Cy5, FAM, and NED=0.100. EGFRvIII RT-PCRcurves were obtained and Ct, delta Ct (dCt), and dRN values werecalculated, and linearity plots were produced.

Plots of data were evaluated to assess the sensitivity of the assays tomeasure total EGFR and EGFRvIII expression in RNA samples prepared fromthe U87vIII EGFRvIII mRNA positive control (see FIG. 16, FIG. 17, andFIG. 18) and to evaluate the sensitivity of the assays to measure totalEGFR expression in RNA samples prepared from the A549 EGFR mRNA positivecontrol (see FIG. 19, FIG. 20, and FIG. 21). Data were assessed withoutrelating it to one of the ACTB, ABL, or G6PD internal controls (Ctvalues in FIG. 16A, FIG. 17A, FIG. 18A, FIG. 19A, FIG. 20A, and FIG.21A) and in relation to one of the ACTB, ABL, or G6PD internal controls(dCt values in FIG. 16B, FIG. 17B, FIG. 18B, FIG. 19B, FIG. 20B, andFIG. 21B).

In addition, the A549 EGFR mRNA control acts as a negative control forthe detection of EGFRvIII. Accordingly, the data collected during theexperiments described herein demonstrates the specificity of theEGFRvIII PCR signal in the methods and experiments described above.

Further, plots of data were evaluated to assess the linearity of theresponse of the measured Ct and dCt values for total EGFR and EGFRvIIIconcentrations with the total RNA in the sample. Plots were preparedcomprising measured Ct values for detection of total EGFR, EGFRvIII, andan internal control (e.g., ACTB, ABL, or G6PD) as a function of thetotal concentration of RNA prepared from the EGFRvIII positive controlU87vIII (FIG. 16A, FIG. 17A, FIG. 18A). The Ct data were used tocalculate dCt values for detection of EGFR and EGFRvIII relative to theinternal control in the sample (FIG. 16B, FIG. 17B, FIG. 18B). Plotswere also prepared comprising measured Ct values for detection of totalEGFR and an internal control (e.g., ACTB, ABL, or G6PD) as a function ofthe total concentration of RNA prepared from the total EGFR positivecontrol A549 (FIG. 19A, FIG. 20A, FIG. 21A). The Ct data were used tocalculate dCt values for detection of total EGFR relative to theinternal control in the sample (FIG. 19B, FIG. 20B, FIG. 21B).

None of the internal controls altered the linearity or sensitivity ofEGFRvIII or EGFRwt detection (e.g., compare FIG. 16A with FIG. 16B, FIG.17A with FIG. 17B, FIG. 18A with FIG. 18B, FIG. 19A with FIG. 19B, FIG.20A with FIG. 20B, and FIG. 21A with FIG. 21B). Amongst the threeinternal controls, beta-actin provided lower Ct values, higher dRNvalues, and a greater sensitivity of detection (e.g., approximately 0.5picograms or more).

The following settings were used for Ct threshold: FAM=0.1, NED=0.75,Cy5=0.1. To determine the relative level of total EGFR mRNA in a givensample, the Ct value of total EGFR is subtracted from the Ct value ofACTB or other internal control to provide a EGFR dCt. Higher EGFR dCtvalues indicate higher EGFR mRNA expression. Similarly, the relativelevel of EGFRvIII mRNA is calculated by subtracting the Ct value ofEGFRvIII from the Ct value of ACTB or other internal control to providea EGFRvIII dCt. Higher EGFRvIII dCt values indicate higher EGFRvIIIexpression. A cutoff dCt can be assigned for EGFRvIII where any EGFRvIIIdCt higher than the cutoff is considered EGFRvIII positive.Alternatively, a maximum EGFRvIII Ct value can be established wherelower (earlier) Ct values indicate EGFRvIII positive. A cutoff dCt canbe assigned for total EGFR where any EGFR dCt higher than the cutoff isconsidered to be a high expresser of EGFR.

Example 4—Evaluation of Linearity and Dynamic Range

During the development of embodiments of the technology provided herein,experiments were conducted to assess the dynamic range and linearresponse of the real-time PCR assay signal to the level of EGFRvIII,tEGFR, and actin expression. Data were collected in real-time PCR assaysof RNA dilution panels prepared by diluting RNA from FFPE cell pelletsfrom a cell line (U87MG de2-7) that expresses EGFRvIII, tEGFR, andactin.

Methods

During the development of embodiments of the technology, experimentswere conducted to detect EGFRvIII mRNA and total EGFR mRNA. In someexperiments, mRNA samples were prepared from formalin-fixedparaffin-embedded (FFPE) cell pellets from a cell line (U87MG de2-7)that expresses EGFRvIII, tEGFR, and actin. In some embodiments, theassays were performed on a commercial real-time PCR instrument such as,e.g., the ABBOTT M2000 REALTIME real-time PCR system. In someembodiments, the assays are used for target amplification, detection ofEGFRvIII target, and relative quantification of total EGFR RNA (vs. anendogenous control gene) either as a complete cocktail or in a subset ofprimer/probe combinations. The compositions described in this examplefind use for amplification of EGFRvIII, total EGFR, and actin mRNA.

Data were collected from samples processed using a manual samplepreparation procedure and sample preparation kit (e.g., an ABBOTTTARGETPREP RNA PRO nucleic acid sample preparation kit). Purified sampleribonucleic acid (RNA) was combined in a 96-well optical reaction platewith an EGFR amplification master mix using a manual process. Theoptical reaction plate was manually transferred to the real-time PCRinstrument for amplification and detection. Assay results wereautomatically reported on the real-time PCR instrument at runcompletion.

Each sample was assayed by reverse transcription (RT) PCR to detectexpression of total EGFR, beta-actin mRNA, and EGFRvIII mRNA. Softwareparameters used on the real-time PCR instrument were provided in anassay application specification file loaded onto the real-time PCRinstrument, e.g., from an assay CD-ROM disk.

Compositions (e.g., reaction mixtures) used for amplification in someembodiments are provided in Table 12 and Table 13 below.

TABLE 12 amplification reagent formulation Final Concentration ReagentComponent Oligo name SEQ ID NO: in 50 μL reaction Unit OligonucleotideEGFRvIII Forward Primer EGvIIIi1_−58to−41 2 0.063 μM Reagent EGFRvIIIReverse Primer EGvIII-A 33 0.282 μM EGFRvIII probe EGvIIIi1_−39to- 420.188 μM −39to−21pFAM-BHQ1 dT Total EGFR Forward EGwti29_−47to−25 80.050 μM Primer Total EGFR Reverse Primer EGwti29_+23to−3 7 0.075 μMTotal EGFR Probe Total EGFR probe 32 0.350 μM beta-Actin (ACTB) ForwardbActl1_−53to−33 14 0.100 μM Primer beta-Actin (ACTB) ReversebActl1_+17to−5 13 0.450 μM Primer beta-Actin (ACTB) probebActl1_−29to−10pNED 43 0.110 μM PCR EZ Buffer N/A N/A 1X Aptamer N/A N/A0.200 μM ROX Reference N/A N/A 0.060 μM dNTPs N/A N/A 0.488 mMActivation MnCl₂ N/A N/A 4.00 mM reagent Enzyme Reagent rTth PolymeraseN/A N/A 15.04 units/ reaction

The PCR EZ Buffer indicated in Table 12 comprises bicine and glycerol.The Oligonucleotide Reagent and Activation Reagent were prepared inmolecular grade water and contain a preservative biocide reagent (e.g.,PROCLIN 300 preservative and/or PROCLIN 950 preservative).

In some embodiments, the primers and probes of the amplification reagentformulation in the “Component” column of Table 12 are the followingprimers and probes in Table 13:

TABLE 13 amplification reagent formulation primer and probe sequencesMaterial Oligo name SEQ ID NO Sequence (5′ to 3′) and descriptionvIII Forward EGvIIIi1_-58to-41  2 CTC CTG GCG CTG CTG GCT PrimervIII Reverse EGvIII-A 33 CGT GAT CTG TCA CCA CAT AAT TAC CTT TC PrimervIII probe (FAM) EGvIIIi1_-39to-21pFAM- 42FAM-CGC TCT GCC CGG CGA GTC G-BHQ1dT BHQ1 dTBHQ1 = Black Hole Quencher 1 Total Forward EGwti29_-47to-25  8CGC CTT GAC TGA GGA CAG CAT AG Primer Total Reverse EGwti29_+23to-3  7GGG AAC GGA CTG GTT TAT GTA TTC AG Primer Total Probe Total EGFR probe32 VIC-A7A C76 67C 67C 7AG 6G-BHQ2dT (VIC) 6 = 5-Propynyl dU7 = 5-Methyl dC BHQ2 = Black Hole Quencher 2 ACTB ForwardbActl1_-53to-33 14 GAG CAC AGA GCC TCG CCT TTG Primer ACTB ReversebActl1_+17to-5 13 TCA TCA TCC ATG GTG AGC TGG C Primer ACTB probebActl1_-29to-10pNED 43 NED-ATC CGC CGC CCG TCC ACA CC-BHQ2dT (NED)

In some embodiments, the “Total EGFR probe” (e.g., of Table 12) has asequence of one of the following oligonucleotides provided in Table 14:

TABLE 14 exemplary sequences of total EGFR probe oligo name short nameoligo type sequence (5′ to 3′) SEQ ID NO: EG129_-21to-5p1 Totalp1 probeVIC-ACA CCT TCC TCC CAG TG-BHQ2dt 38 EG129_-21to-5p2 Totalp2 probeVIC-A7A C76 67C 67C 7AG 6G-BHQ2dt 39 EG129_-21to-5p3 Totalp3 probeVIC-A5A C56 65C 65C 5AG 6G-BHQ2dt 40 EG129_-21to-5p4 Totalp4 probeVIC-A7A C56 67C 65C 5AG 6G-BHQ2dt 41

In Table 14, a base indicated by a “5” is a 5-Propynyl dC, a baseindicated by a “6” is a 5-Propynyl dU, and a base indicated by a “7” isa 5-Methyl dC. In Table 14, BHQ2 indicates Black Hole Quencher 2 and VICindicates a VIC fluorescent moiety.

In some embodiments, the amplification cycling conditions used in thereal-time PCR assays described herein are those provided in Table 15:

TABLE 15 amplification cycling conditions Step Number of Number CyclesDescription Temperature Time 1 1 Denaturation 95° C. 1 minute 2 1Reverse Transcription 62° C. 30 minutes 3 4 Denaturation 92° C. 30seconds Annealing 60° C. 30 seconds 4 50 Denaturation 92° C. 30 secondsAnnealing 62° C. 30 seconds Extension 58° C. 40 seconds

During the development of some embodiments of the technology, data werecollected to evaluate the linearity and dynamic range of the EGFR assay(e.g., using reaction mixtures as described above). For example,experiments were conducted in which the linearity and dynamic range wereevaluated using RNA dilution panels prepared by diluting RNA from FFPEcell pellets from a cell line (U87MG de2-7) that expresses EGFRvIII,tEGFR, and actin. The RNA was diluted in molecular biology grade waterto prepare dilution panels ranging from 9320 pg to 0.0022 pg RNA per PCRwell. Three PCR replicates of each dilution panel on each of threereal-time PCR instruments were tested, resulting in a total of 9replicates. Real-time data collected for EGFRvIII, total EGFR, and actinare shown in FIGS. 23, 24, and 25, respectively.

Results

The dynamic range and limit of quantification were evaluated. Inparticular, the dynamic range was calculated as the range of RNA amountdemonstrating 100% detection of replicates with <5% CV. The limit ofquantification was calculated as the cycle number value of the lowestdilution panel member within the dynamic range. Results are provided inTable 16:

TABLE 16 Dynamic range and Limit of Quantitation (LoQ) cycle Limit ofnumber Quantification (LoQ) Target Dynamic Range range (CN) EGFRvIII9320 pg to 0.569 pg 18.86-34.63 34.63 Total 9320 pg to 0.284 pg15.79-33.46 33.46 EGFR Actin 9320 pg to 0.142 pg 17.46-33.70 33.70

The data collected indicated that the RNA extracted from FFPE cellpellets from a cell line (U87MG de2-7) that expresses EGFRvIII, tEGFR,and actin provides adequate linearity for EGFRvIII (see FIG. 23), totalEGFR (see FIG. 24), and Actin (see FIG. 25) across a wide range of Ctvalues. EGFRvIII demonstrated dynamic range of 4.21 log (9320 pg to0.569 pg) and a limit of quantitation for EGFRvIII was determined to be34.63 cycles. Total EGFR demonstrated a dynamic range of 4.52 log (9320pg to 0.284 pg) and a limit of quantitation for Total EGFR wasdetermined to be 33.46 cycles. Actin demonstrated dynamic range of 4.82log (9320 pg to 0.142 pg) and a limit of quantitation for actin wasdetermined to be 33.70 cycles.

Example 5—Specificity Study

Further experiments were conducted during the development of embodimentsof the technology disclosed herein. In these experiments, thespecificity of the real-time assay for the EGFRvIII target was assessedby evaluating the EGFRvIII positive or negative status for 20 non-GBMbrain tissue specimens from normal donors. Amplification of EGFRvIIImRNA to generate an EGFRvIII-positive result was not expected for thespecimens in this study, so EGFRvIII negativity rate was used to assessthe analytical specificity for EGFRvIII.

Methods

Three FFPE slide sections for each of the 20 non-GBM brain specimenswere processed for RNA extraction using TargetPrep RNA Pro kit. EGFRmastermix was prepared by combining components of the EGFR amplificationreagent kit. EGFR amplification mastermix configuration is listed inTable 12. The RNA eluates were mixed with EGFR mastermix in wells of a96-well optical reaction plate using a manual process. The opticalreaction plate was manually transferred to the real-time PCR instrumentfor amplification and detection. Assay results were automaticallyreported on the real-time PCR instrument at run completion. For eachspecimen, 3 replicates were tested with 1 lot of EGFR assay reagentsacross 3 runs (1 replicate per run) on a single real-time PCRinstrument.

Results

EGFRvIII positivity and negativity status were reported by comparing theEGFRvIII CN values to a clinical cutoff value of 30.00 CN. EGFRvIIIresult was reported as ‘NEG’ (negative) for specimens with a CNvalue >30.00 or a CN value of −1. Data were collected from allreplicates from all three runs and are presented in Table 17.

TABLE 17 Specificity analysis Number of EGFRvIII Number of negativenegative specimens EGFRvIII Run specimens tested detected NegativityRate 1 20 20 100.0% 2 20 20 100.0% 3 20 20 100.0%

As indicated by the data, the 20 non-GBM brain tissue specimens fromnormal donors did not generate a positive EGFRvIII assay result in anyof the 3 individual runs; therefore, an EGFRvIII Negativity Rate of100.0% was attained. This suggests that EGFRvIII primers and probes areselectively amplifying and detecting EGFRvIII signal and noamplification is observed from non-GBM brain specimens.

Example 6—Testing GBM Specimens

In additional experiments conducted during the development ofembodiments of the technology described herein, thirty-two (32) FFPEglioblastoma multiforme (GBM) specimens were screened using thereal-time assay for detection of EGFRvIII and relative expression oftotal EGFR. EGFRvIII positive or negative, and Total EGFR positive andnegative status, were determined for the set of GBM specimens.

Methods

The FFPE glioblastoma multiforme (GBM) specimens were obtained fromcommercial vendors (Asterand and Discovery Life). Amplification ofactin, total EGFR, and EGFRvIII mRNA were evaluated. During theexperiments, RNA was extracted from one slide from each specimen usingTargetPrep RNA Pro kit and PCR replicatens were tested from each RNAeluate. Sample preparation was performed in 2 batches. In each batch, 1EGFR negative control and 1 EGFR positive control were included. EGFRmastermix was prepared by combining components of the EGFR amplificationreagent kit. EGFR amplification mastermix configuration is listed inTable 12. The RNA eluates were mixed with EGFR mastermix in wells of a96-well optical reaction plate using a manual process. The opticalreaction plate was manually transferred to the real-time PCR instrumentfor amplification and detection. Assay results were automaticallyreported on the real-time PCR instrument at run completion.

Results

The data collected are shown in Table 18. Average cycle numbers from twoPCR replicates of EGFRvIII (“EGFRvIII CN”), total EGFR (“tEGFR CN”), andactin (“ACTB CN”) are reported in Table 18. These data were used tocalculate the total EGFR relative to actin (“tEGFR dCN”) and presence orabsence of EGFRvIII. These results were used to assign a “positive” or“negative” result to the total EGFR and EGFRvIII assays.

TABLE 18 Results from testing FFPE GBM specimens EGFR VIII tEGFR ACTBtEGFR Sample ID Vendor CN CN CN dCN EGFR Result 1183334B Asterand ND21.14 15.49 −5.66 tEGFR NEG; vIII NEG 1174894B Asterand ND 20.17 14.44−5.74 tEGFR NEG; vIII NEG 1199188B Asterand ND 20.61 14.24 −6.37 tEGFRNEG; vIII NEG 1199193B Asterand 34.36 20.17 13.65 −6.52 tEGFR NEG; vIIINEG 1180366B Asterand ND 22.15 15.54 −6.61 tEGFR NEG; vIII NEG R13-0321Discovery ND 27.52 20.88 −6.65 tEGFR NEG; vIII NEG Life R15-0034Discovery ND 24.44 17.16 −7.28 tEGFR NEG; vIII NEG Life 1179610BAsterand ND 19.74 12.40 −7.34 tEGFR NEG; vIII NEG R15-0028 Discovery35.74/ 18.43 11.06 −7.37 tEGFR NEG; vIII NEG Life ND 1180569B AsterandND 23.03 15.40 −7.63 tEGFR NEG; vIII NEG R13-0397 Discovery ND 29.1021.42 −7.69 tEGFR NEG; vIII NEG Life R14-0493 Discovery ND 32.47 24.77−7.70 tEGFR NEG; vIII NEG Life 1199219B Asterand 35.63/ 22.60 14.56−8.04 tEGFR NEG; vIII NEG ND 1184408B Asterand ND 18.18 18.85 0.68 tEGFRPOS; vIII NEG R13-0588 Discovery 33.94 15.88 15.41 −0.47 tEGFR POS; vIIINEG Life 355098B4 Asterand 31.43 20.44 19.63 −0.81 tEGFR POS; vIII NEG388101A1 Asterand 32.25 25.53 24.41 −1.13 tEGFR POS; vIII NEG 1184693BAsterand 30.23 22.17 20.90 −1.28 tEGFR POS; vIII NEG 355101A1 Asterand34.40 23.72 22.30 −1.42 tEGFR POS; vIII NEG 1194912B Asterand ND 22.0119.88 −2.13 tEGFR POS; vIII NEG R15-0002 Discovery ND 17.94 15.63 −2.31tEGFR POS; vIII NEG Life 388085A2 Asterand ND 30.78 27.94 −2.84 tEGFRPOS; vIII NEG 354254B1 Asterand ND 27.94 24.81 −3.14 tEGFR POS; vIII NEG1199198B Asterand 32.05 16.39 13.15 −3.24 tEGFR POS; vIII NEG 388104A1Asterand ND 29.15 24.50 −4.65 tEGFR POS; vIII NEG 1194915B Asterand ND19.35 14.15 −5.20 tEGFR POS; vIII NEG 1197047B Asterand 17.85 14.4612.12 −2.34 tEGFR POS; vIII NEG 1199211B Asterand 18.37 13.11 13.12 0.01tEGFR POS; vIII NEG R14-0593 Discovery 20.76 17.13 13.97 −3.16 tEGFRPOS; vIII NEG Life 1199394B Asterand 21.04 17.00 15.38 −1.62 tEGFR POS;vIII NEG 1180381B Asterand 22.74 14.78 13.41 −1.37 tEGFR POS; vIII NEG1204786B Asterand 27.33 18.10 17.05 −1.05 tEGFR POS; vIII NEG

In Table 18, EGFRvIII CN is reported as “ND” (not detected) if there wasno signal for the EGFRvIII target. For sample IDs R15-0028 and 1199219B,the EGFRvIII signal was detected from only 1 PCR replicate.

Out of the 32 specimens tested in this study, 6 were positive and 26were negative for EGFRvIII. Positive and Negative status for EGFRvIIIwere determined by comparing the EGFRvIII CN values to a clinical cutoffvalue of 30.00 CN. The EGFRvIII result is reported as “POS” (positive)for specimens having a CN value less than or equal to 30.00 and “NEG”(negative) for specimens having a CN value greater than 30.00 or a CNvalue that was not detected. Relative expression of total EGFR for thisstudy was determined by calculating the difference between mean ACT CNand mean tEGFR CN values, which is referred as delta CN (dCN). Delta CNvalues for each specimen were compared with a clinical cutoff value of−5.5. tEGFR results are reported as “POS” (positive) for specimenshaving a delta CN greater than or equal to −5.5 and “NEG” (negative) forspecimens having a delta CN less than −5.5. Out of the 32 specimenstested in this study, 19 specimens were positive and 13 were negativefor total EGFR.

All publications and patents mentioned in the above specification areherein incorporated by reference in their entirety for all purposes.Various modifications and variations of the described compositions,methods, and uses of the technology will be apparent to those skilled inthe art without departing from the scope and spirit of the technology asdescribed. Although the technology has been described in connection withspecific exemplary embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention that are obvious to those skilled in the artare intended to be within the scope of the following claims.

We claim:
 1. A kit for detecting EGFRvIII mRNA, comprising a primercomprising a sequence according to SEQ ID NO: 1 or SEQ ID NO: 31, aprimer comprising a sequence according to SEQ ID NO: 2, and a detectablylabeled probe comprising a sequence according to SEQ ID NO:
 3. 2. A kitfor detecting EGFRvIII mRNA and for quantifying total EGFR mRNA,comprising a primer comprising a sequence according to SEQ ID NO: 1 orSEQ ID NO: 31, a primer comprising a sequence according to SEQ ID NO: 2,a primer comprising a sequence according to SEQ ID NO: 7, a primercomprising a sequence according to SEQ ID NO: 8, a detectably labeledprobe comprising a sequence according to SEQ ID NO: 3, and a detectablylabeled probe comprising a sequence according to SEQ ID NO: 9 or SEQ IDNO:
 32. 3. The kit of claim 2, further comprising a primer comprising asequence according to SEQ ID NO: 13, a primer comprising a sequenceaccording to SEQ ID NO: 14, and a detectably labeled probe comprising asequence according to SEQ ID NO:
 15. 4. The kit of claim 3, wherein thedetectably labeled probe comprising a sequence according to SEQ ID NO: 3comprises a first distinguishable fluorescent moiety, the detectablylabeled probe comprising a sequence according to SEQ ID NO: 9 or SEQ IDNO: 32 comprises a second distinguishable fluorescent moiety, and thedetectably labeled probe comprising a sequence according to SEQ ID NO:15 comprises a third distinguishable fluorescent moiety.
 5. A system fordetecting EGFRvIII mRNA, comprising a primer comprising a sequenceaccording to SEQ ID NO: 1 or SEQ ID NO: 31, a primer comprising asequence according to SEQ ID NO: 2, and a detectably labeled probecomprising a sequence according to SEQ ID NO:
 3. 6. A system fordetecting EGFRvIII mRNA and for quantifying total EGFR mRNA, comprisinga primer comprising a sequence according to SEQ ID NO: 1 or SEQ ID NO:31, a primer comprising a sequence according to SEQ ID NO: 2, a primercomprising a sequence according to SEQ ID NO: 7, a primer comprising asequence according to SEQ ID NO: 8, a detectably labeled probecomprising a sequence according to SEQ ID NO: 3, and a detectablylabeled probe comprising a sequence according to SEQ ID NO: 9 or SEQ IDNO:
 32. 7. The system of claim 6, further comprising a primer comprisinga sequence according to SEQ ID NO: 13, a primer comprising a sequenceaccording to SEQ ID NO: 14, and a detectably labeled probe comprising asequence according to SEQ ID NO:
 15. 8. The system of claim 7, whereinthe detectably labeled probe comprising a sequence according to SEQ IDNO: 3 comprises a first distinguishable fluorescent moiety, thedetectably labeled probe comprising a sequence according to SEQ ID NO: 9or SEQ ID NO: 32 comprises a second distinguishable fluorescent moiety,and the detectably labeled probe comprising a sequence according to SEQID NO: 15 comprises a third distinguishable fluorescent moiety.
 9. Amethod for treating a patient with an anti-EGFR agent wherein thepatient is is suffering from cancer, the method comprising the steps of:a) determining or having determined the presence of EGFRvIII expressionin a sample by: 1) obtaining a biological sample from a patient; 2)mixing the patient sample with a composition comprising a primercomprising a sequence according to SEQ ID NO: 1 or SEQ ID NO: 31, aprimer comprising a sequence according to SEQ ID NO: 2, and a detectablylabeled probe comprising a sequence according to SEQ ID NO: 3 to providea RT-PCR reaction mixture; 3) reverse transcribing EGFRvIII mRNA toprovide a EGFRvIII cDNA; 4) amplifying the EGFRvIII cDNA to provide anEGFRvIII amplicon; and 5) detecting the EGFRvIII amplicon, whereindetecting the EGFRvIII amplicon indicates the presence of EGFRvIIIexpression in the sample; and b) treating the patient with ananti-EGFRvIII agent if EGFRvIII expression is present.
 10. The method ofclaim 9, wherein said anti-EGFRvIII agent is an anilinoquinazoline,lapatinib, and/or an antibody.
 11. The method of claim 9, wherein saidanti-EGFRvIII agent is Vandetanib (Caprelsa), Panitumumab (Vectibix),Gefitinib (Iressa), Erlotinib (Tarceva), and/or Afatinib (Gilotrif). 12.The method of claim 9, wherein said anti-EGFRvIII agent comprises atherapy targeting signaling downstream of EGFR comprising a therapytargeting the genes or the products of genes encoding KRAS, BRAF, MEK,ERK, PI3K, phospholipase C gamma, AKT, and/or STAT.
 13. The method ofclaim 9, wherein said anti-EGFRvIII agent comprises a combinationtherapy comprising treating a subject with an anti-EGFR therapeuticagent and one or more of a radiation therapy or a chemotherapy.
 14. Themethod of claim 9, wherein said anti-EGFRvIII agent comprises aplatinum-based therapy, a topoisomerase inhibitor, and/or a taxane. 15.The method of claim 9, wherein said anti-EGFRvIII agent comprises adietary bioactive agent.
 16. The method of claim 9, wherein said dietarybioactive agent comprise capsaicin, genistein, or curcumin.
 17. Themethod of claim 9, wherein said anti-EGFRvIII agent comprises anantibody that recognizes EGFR.
 18. The method of claim 17, wherein saidantibody that recognizes EGFR comprises a monoclonal antibody and/or apolyclonal antibody.
 19. The method of claim 17, wherein said antibodyis an antibody drug conjugate.
 20. The method of claim 9, wherein stepsa) and b) are repeated.